FEMS Microbiology Reviews 15 (1994) 321-353 (c,~ 1994 Federation of European Microbiological Societies I)168-6445/94/$26.00 Published by Elsevier 321 F E M S R E 011439 Flemming Eke|und * and Regin R~ann l)epartment of Population Biolog3, Zoologieal Institute, Unit,ersity of Copenhagen, Uniz,ersitetsl~arken 15, DK-2100 Copenhagen, Denmark (Received 8 October 1993: accepted 18 May 1994) Abstract." Heterotrophic flagellates and naked amoebae are usually very numerous in agricultural soils; with numbers in thc magnitude of 10000 to 100000 (active + encysted) cells per gram of soil. In "hotspots' influenced by living roots or by dead organic material, the n u m b e r may occasionally be as high as several millions per gram of soil. An exact enumeration of these organisms is virtually impossible. As they most often adhere closely to the soil particles, direct counting will underestimate numbers since the organisms will be masked. The method usually applied for enumeration of these organisms, the 'most probable number (MPN) method', is based on the ability of the organisms to grow on particular culture media. This method will in many cases underestimate the total protozoan number (active + encysted). It is uncertain how many of the heterotrophic flagellates and naked amoebae are actively moving and how many are encysted at a particular time: the 'HCl-method" which has usually been used to discriminate between active and encysted has proven to be highly unreliable. Despite the methodological difficulties many investigations of these organisms indicate that they play an important role in agricultural soils as bacterial consumers, and to a minor extent as consumers of fungi. Because of their small size and their flexible body they are able to graze bacteria in small pores in the soil in which larger organisms are precluded from coming. Key factors restricting the n u m b e r and activity of heterotrophic flagellates and naked amoebae in soils seem to be water potential and soil structure and texture. In micro-cosm experiments, small heterotrophic flagellates and naked amoebae regulate the size and composition of the bacterial community. Bacterial activity seems to be stimulated by these organisms in most cases as well as the mineralization of carbon and nitrogen and possibly other mineral nutrients. In the rhizosphere of living plants the activity of protozoa has proven to stimulate uptake of nitrogen in pot experiments, and it has been hypothesized that organic matter liberated by plants in the root zone will stimulate bacterial and protozoan activity. leading to mineralization of organic soil nitrogen which is subsequently taken up by the plants. Key, words." Soil protozoa: Naked amoebae: Heterotrophic flagellates: Detrital food web; Mineralization; Rhizosphere * Corresponding author. Tel.: ( + 46-3532) 1275; Fax: (+45-3532 1300; e-mail: FEkelund(r~ZI,KU.I)K SSD1 01 68-6445194)01)1141 -V Downloaded from https://academic.oup.com/femsre/article/15/4/321/489287 by guest on 12 July 2022 Notes on protozoa in agricultural soil with emphasis on heterotrophic flagellates and naked amoebae and their ecology 322 Contents 321 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T h e study of soil p r o t o z o a . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The soil p r o t o z o a n c o m m u n i t y . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 323 323 323 Q u a l i t a t i v e study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Isolation a n d c u l t u r e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 324 324 I d e n t i f i c a t i o n of h e t e r o t r o p h i c flagellates a n d n a k e d a m o e b a e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 324 P a t h o g e n i c soil a m o e b a e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325 F u n d a m e n t a l biology a n d ecology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Life cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325 325 T h e cyst . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325 Nutrition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Osmotrophy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32(~ 327 Phagotrophic feeding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 327 E n u m e r a t i o n of soil p r o t o z o a , p r o t o z o a n n u m b e r s a n d b i o m a s s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32~ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32L~ A c t i v e vs. non active soil p r o t o z o a : T h e "HCI m e t h o d " . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T h e direct c o u n t i n g m e t h o d . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E n u m e r a t i o n of soil p r o t o z o a : the M P N m e t h o d 330 33(I T h e n u m b e r a n d b i o m a s s of p r o t o z o a in a r a b l e soil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 331 T h e effect of p h y s i c a l / / c h e m i c a l factors on soil p r o t o z o a . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333 Soil Soil Soil Soil protozoa and pH . . . . . . . . . . . . . . . protozoa and t e m p e r a t u r e . . . . . . . . . p r o t o z o a vs. o x y g e n / c a r b o n dioxide . . . . p r o t o z o a : soil w a t e r , texture a n d s t r u c t u r e . . . . 333 334 335 335 T h e ecological role of the soil p r o t o z o a . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33~+ T h c p r o t o z o a in the detrital food w e b . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T h e effect of p r o t o z o a n g r a z i n g on bacterial p o p u l a t i o n s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . R e g u l a t i o n of thc size o f bacterial p o p u l a t i o n s 33~'+ 337 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 337 Effects of p r o t o z o a on c o m p o s i t i o n of microbial c o m m u n i t i e s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S t i m u l a t i o n of bacterial activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 338 33~) Effect of p r o t o z o a on fungal p o p u l a t i o n s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Effect of p r o t o z o a n g r a z i n g on d e c o m p o s i t i o n a n d m i n e r a l i z a t i o n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T h e use of soil m i c r o c o s m s in studies of m i n e r a l i z a t i o n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33t~ 33 t) 340 Effect on C - m i n e r a l i z a t i o n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Effect on N - m i n e r a l i z a t i o n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 340 332 Effect on m i n e r a l i z a t i o n of o t h e r n u t r i e n t s . . . . . . . . . . . . . . . . . . . . . . . . . . . . P r e d a t o r s a n d p a r a s i t e s on soil p r o t o z o a . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . P r o t o z o a in a r a b l e soil a n d t h e i r relation to plant g r o w t h . . . . . . . . . . . . . . . . . . . . . . . P r o t o z o a in the r h i z o s p h e r e . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T h e r h i z o s p h e r c effect . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Effects of p r o t o z o a on plant growth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ( ' l a r h o l m ' s hypothesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . O t h e r i n t e r a c t i o n s b e t w e e n p r o t o z o a and plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Important literature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Rctercnccs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 342 342 343 343 343 344 ~45 345 3&'~ 34~ Downloaded from https://academic.oup.com/femsre/article/15/4/321/489287 by guest on 12 July 2022 Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 323 Introduction The study of soil protozoa The soil protozoan community Four different types of protozoa are frequently found in soil: ciliates, heterotrophic flagellates, naked amoeba, and testate amoebae. Other types are very rarely found, e.g. the heliozoan genus Actinomonas [20] that has been reported a few times from soil [21]. Of these only the ciliates can be considered a natural taxonomic unit [22]. Although testate amoebae do not constitute a natural taxonomic unit, they are usually consid- [13]. The borderline between flagellates and amoebae is not sharp. The typical flagellate is a swimming organism, while the typical amoebae uses its pseudopodia to 'crawl' around on the soil particles. However, some of the amoebae, the amoeboflagellates, posses flagella at some stages of their life cycles and many soil flagellates have capabilities of pseudopodia formation or amoeboid movement (e.g. Cercomonas). Traditionally the soil fauna has been classified in microfauna, mesofauna, and macrofauna [27]. However, small soil flagellates and amoebae could be termed 'the nanofauna', analogous to nanoplankton [28]; these organisms are able to enter pore necks as small as 3 p.m, and are able to consume bacteria and fungi in narrow spaces where larger predators are precluded from entering. Though heterotrophic flagellates and naked amoebae constitute a very important part of the soil food web, ciliates and testate amoebae have gained more attention, probably because they are larger and therefore easier to handle and examine. The two last-mentioned groups have been reviewed excellently by Foissner [18], where numerous references to literature on general soil protozoological subjects can be found, this paper should be consulted not least for its covering of Russian and East-European literature. Below we will focus mainly on heterotrophic flagellates and naked amoebae and as a rule only make corn- Downloaded from https://academic.oup.com/femsre/article/15/4/321/489287 by guest on 12 July 2022 The study of soil protozoa started in the early part of this century, stimulated by the hypothesis by Russell and Hutchinson [1] that protozoa make the soil less fertile by reducing the number of bacteria. At first it was discussed whether active protozoa occur in soil, or if they are present only as cysts; this controversy was settled, however, when the work of Martin and Lewin [2] definitely showed that active protozoa do occur in soil. Studies carried out at Rothamsted Experimental Station demonstrated that protozoa play an important role in the regulation of bacterial populations [3,4], but also that the effect was not deleterious. On the contrary, it stimulated bacterial activity in many cases (e.g. [5]). Extensive research on differential protozoan predation was done by Singh [6]. Darbyshire and Greaves [7] made extensive work on rhizosphere protozoa. Later on, in the late seventies protozoan grazing on bacterial populations and the subsequent release of mineral nutrients have increasingly been an object of interest. Pioneering work in this field has been done by workers from Fort Collins, Colorado, e.g. [8-12]. The effects on plants of bacterial grazing by protozoa have later been studied by Clarholm [13,14] and Kuikman [15]. Other subjects, lately brought into focus in protozoan research, are mycophagous amoebae and their possible role in controlling plant diseases (e.g. [16,17]), autecology of ciliates and testate amoebae [18], and the effect of protozoa on bacteria introduced into soil [19]. ered to make up an ecological unity [23]. Testate amoebae have a long generation time, reproducing only about once a week, and can be considered to be K-strategists compared to the other groups [24]. Many testate amoebae may be primary decomposers; some species have been observed to ingest detrital particles when bacteria were less available [25]. Most naked amoebae, flagellates, and ciliates feed on bacteria; these groups are generally abundant in agricultural soil [23]. Ciliates are, generally, far less numerous in soil than flagellates and amoebae [26]. Because of their large size compared to the flagellates and amoebae (table 1), they are probably more abundant in wet soils where relatively large pores arc water-filled 324 Qualitative study The easiest way to observe soil protozoa is in an enrichment culture made by placing a small portion of soil in a petridish and soak it with water or some weak salt solution. The Petri dish is incubated in darkness at room temperature, and protozoa can usually be observed already after a few hours; the community will change with time and new forms can be observed by regular inspection the next 1-2 months. The protozoa can be observed directly in the soil free part of the Petri dish, or in a drop of water transferred to a glass slide. Further details on enrichment cultures are given by Page [31,32] and by Foissner [18], The dilution technique for enumerating soil protozoa mentioned below (see below) can also be applied for qualitative studies. As a by-product of the estimation of the soil protozoan number, enrichment cultures, often pure, of the more common soil protozoa are produced in the high dilutions. In our experience the dilution method yields small forms while the 'Petri dish method' mainly produces larger forms (R0nn and Ekelund, unpublished data). Isolation and culture In most cases it will be necessary to isolate and establish pure cultures of naked amoebae and heterotrophic flagellates prior to a closer study and possible identification. Identification of single specimens is as a rule not possible, except for very characteristic forms. Two methods of isolation are commonly applied. By the micro-manipulatory method, individual ceils are transferred manually from enrichment cultures to some culture media by fine microcapillaries. The process is best carried out under an inverted microscope. This method has to be applied if the studied organism is rare or grows poorly in enrichment culture, but requires some skill. The serial dilution method can be used on numerically abundant organisms. Small amounts of liquid from culture suspensions are transferred repeatedly to fresh media, until the original culture is sufficiently diluted, and only a single or a few cells are left. The original inoculum can be either an enrichment culture or a suspended soil sample. The dilution factor used may vary from 2 to 10. When the dilution method is used to enumerate soil protozoa (see below), pure cultures are often produced as a by-product (see also [31,331). Many organisms will do well on the bacteria accompanying them from nature, on a weak nutrient medium these food bacteria will multiply. Alternatively, protozoa can be grown on a nonnutrient medium where bacteria of an identified strain are added. Some larger amoebae seem unable to live on bacteria alone, and must be supplied with other amoebae, flagellates, or algae. Further information on culture methods is given by [32] and [34]. Identification of heterotrophic flagellates and naked amoebae Identification of heterotrophic soil flagellates (and probably heterotrophic flagellates in general) by routine is virtually impossible, even for experts: updated comprehensive literature does not exist. Since Pascher [35] and L e m m e r m a n n [36], no assembled work for identifying flagellates has been published. H~inel's [37] key to heterotrophic flagellates in sewage will sometimes yield useful results, and identification to the generic level can sometimes be achieved by the method of Patterson and Hedley ([38]; and references therein). A large number of synonyms exist both at the specific and at the generic level, and there seem to be several undescribed species Downloaded from https://academic.oup.com/femsre/article/15/4/321/489287 by guest on 12 July 2022 mcnts on testate a m o e b a e and ciliates in connection with subjects of general protozoological interest or when no data on naked amoebae and heterotrophic flagellates are available. The sporulating groups of naked amoebae are omitted; this more a matter of convenience than based on taxonomical or ecological reasons. The amoebal stages in the life cycles of slime moulds resemble other naked amoebae both in appearance and ecology. More information about the ecology of these organisms is given in refs. [29,30]. 325 Pathogenic soil amoebae People working with soil protozoa should be aware that some strains of free-living amoebae may be pathogenic. Naegleria fowleri (= N. aerobia of Singh) may cause lethal meningitis [42,43]. The amoebae probably enter the central nervous system by penetrating the nasal mucosa and migrate to the brain along the olfactory nerves. However, the pathogenic strains probably only grow at relatively high temperatures and not at room temperature (20-22 °C) [32]. Furthermore the cyst of N. fowleri is not resistant to extreme drying [42,43], and it is therefore unlikely that this species is common in soil. Most of the infections reported are believed to be caused by bathing in warm ponds or heated swimming pools, in which conditions for growth of these amoebae are favourable [42]. Some strains of Acanthamoeba are also pathogellic and may infect the central nervous system in a manner similar to Naegleria [42,44], or infect the eye [32,42]. Some of these strains are common in soils [43] and as they also grow at room temperature [32] the risk of obtaining them in enrichment cultures from soil are larger than is the case with Naegleria. Still, there are no reports of infections obtained during laboratory work (J.F. De Jonckheere, Instituut voor Hygiene en Epidemiologie, Brussels, Belgium (1992) personal communication). Fundamental biology and ecology Life cycle Most of the organisms treated here have simple life cycles, including only an active, trophic stage, and a resting stage: the cyst. The active stage is often called the trophozoite, but Margulis et al. [34] use this term only for motile trophic stages of biotrophic protists. In addition to the amoebal and cystic stages, a few amoebae, the so-called amoeboflagellates, also have a flagellate stage in their life cycle. Sexuality seems to be included in the life cycle of heterotrophic flagellates and amoebae only as an exception, but, as Fenchel [46] points out, absence of sexuality is not easy to establish with certainty, since it may be quite difficult to observe in a given group. Sexuality has only rarely been reported among amoebae. Until recently there were only poorly documented reports of sexual fusion in the testate species [47,48], but Mignot and Raikov [49] definitely demonstrated the existence of meiosis in the testate amoebae Arcella t'ulgaris. Recently meiosis has also been observed in a vampyrellid amoeba [50]. Among the heterotrophic flagellates, sexuality has been observed in the Dinoflagellates [51], which rarely occur in soil, and in some heterocont monads [52]; but whether it is found among the heterocont soil flagellates is not known. The Euglenids are most often considered an asexual group, even though sexuality has been reported once [53]. Thus nearly all soil flagellates must be considered asexual organisms at present. The cyst Most soil protozoa have the ability to encyst. The cyst is an isodiametrical, spherical or polyhedron shaped structure; in most cases it is formed Downloaded from https://academic.oup.com/femsre/article/15/4/321/489287 by guest on 12 July 2022 even among the most commonly occurring forms. In most cases, it is necessary to use EM techniques on pure cultures. Uncertain identifications ought to be checked by a specialist since doubtful recordings are almost worthless. Taxonomy of heterotrophic flagellates is covered by Patterson and Larsson [39]. As opposed to the situation concerning the heterotrophic flagellates, updated literature for identification of naked amoebae does exist. Amoebae with lobose pseudopodia are covered by the excellent key by Page [32]. If the pseudopodia are of the filose type, or if they form anastomosing networks, the organism in question belong to one of the groups not covered in this key, but they are included in the key by Page and Siemensma [40]. Identification by routine to at least generic level by normal light microscopic techniques is possible in many cases but not all. Identification to species level is in most cases very difficult. The taxonomy of naked amoebae is covered by Page [41]. 326 stances produced as a result of protein degradation can cause excystment [58]. Some workers have focused more on the role of carbon dioxide in excystment of amoebae. Averner and Fulton [61] showed that slightly increased environmental carbon dioxide content caused excystment in Naegleria gruberi. Addition of proline or an increased cyst density also caused excystment, but these factors had effect only by increasing the carbon dioxide level; the authors therefore suggested that carbon dioxide was the only signal for excystment in Naegleria gruberi. However, Singh et al. [62] showed that aqueous extracts of bacteria caused excystment of Vahlkampfia (Schizopyrenus) russelli in a bacteria-free environment, and that some amino acids and nucleotides were the active components. Datta [63] showed that an atmospheric level of carbon dioxide was necessary for excystation of Vahlkampfia (Schizopyrenus) russelli and Acanthamoeba (Hartmanella) rhysodes, and that levels up to 0.8% enhanced the process, whereas levels from 1.0 to 3.0% were inhibitory. Excystation did not occur in distilled water but most salts of sodium, copper, and potassium and Escherichia coli extract induced excystation, while salts of ammonium had little or no effect. Soil protozoan activity will most often be confined to short periods with beneficial conditions and excystation will, as a rule, occur quickly in response to e.g. rewetting or thaw. Clarholm [64] showed that large amoebal populations were built up shortly after rainfall, and Hughes and Smith [65] showed that given the proper conditions the common soil flagellate Heteromita globosa was able to ex- or encyst in about 1 h. Testate amoebae only excyst in response to rewetting after several days and the period needed for excystment increases with the duration of the previous period of dryness [66]; these observations probably reflect an adaptation to the special mode of life of the testate amoebae (K-strategy) and are probably not valid for fast-growing forms, like many small flagellates and naked amoebae. Nutrition Heterotrophic protozoa have to obtain their organic carbon and energy supplies from the en- Downloaded from https://academic.oup.com/femsre/article/15/4/321/489287 by guest on 12 July 2022 as a protection against adverse conditions. It may, however, also serve other purposes, e.g. dispersal. Kingston and Warhurst [54] showed that in outdoor air there was one amoebal cyst in each 9.1 m 3 air, and Lawande [55] isolated several species of soil a m o e b a e from air during dust storms in Nigeria. Cysts will often pass undigested through the gut of soil animals and may in this way be transported in the soil [56]. Most papers on protozoan cysts deal with ciliates, whereas less is published on amoebae, and very little on flagellates. Corliss and Esser [57] have given a short review of the role of the cyst in free-living protozoa, and Wagtendonk [58] has given a more detailed review on encystment and excystment. Although we mainly consider flagellates and amoebae in this work, we will here refer to some general statements about cysts that are based on experiments with ciliates. The conclusions reached about the factors leading to encystment are various and often contradictory. A key factor in causing encystment seems to be the deficiency of food, or deficiency of specific growth factors such as vitamins. However, some organisms seem to encyst only under optimum growth conditions when food is in excess. Crowding may also be an important factor as well as bacterial and protozoan excretion products. Abiotic factors that cause encystment include desiccation, increased salt concentration, extreme p H values, and lack of oxygen [57,58]. Desiccation is a special problem for terrestrial protozoa, and they are generally able to produce desiccation-resistant cysts in response to evaporation of the surrounding water. Goodey [59] found that both flagellates and amoebae had survived in soil that had been air-dried and subsequently stored for 47 years. Cysts can also be resistant to extreme temperatures; Rogerson and Berger [60] found that active forms rapidly appeared in garden soil that had been stored at - l l ° C for 1 year. Colpoda cysts are able to withstand wet heat up to 50°C and dry heat up to 120°C [46]. Logically, excystment is to be expected when the environmental conditions are favourable and therefore it is not surprising that factors like osmotic potential, adequate food supply, oxygen concentration and the presence of organic sub- 327 vironment, either by absorbing organic substances over the cell m e m b r a n e (osmotrophy) or by ingesting particulate organic material by phagocytosis. Osmotrophy Phagotrophic feeding It is generally accepted that the main food of the soil protozoa is bacteria, though a wide range of other food objects are also utilized. Of 258 species of protozoa (63 flagellates, 98 amoebae and 97 ciliates) from the rhizosphere of plants, 40% were believed to be strict bacterial feeders and 36% of the remaining species had bacteria as an important part of their diet. Twenty percent lived completely or partly of other protozoa. Fungi, detritus and algae were eaten by a smaller number of protozoa [71]. A. Bacterivory. Bacteria differ in their suitability as food. Experiments with a m o e b a e grown monoxenically with different species of bacteria have shown that some species are readily eaten Downloaded from https://academic.oup.com/femsre/article/15/4/321/489287 by guest on 12 July 2022 Many soil protozoa, as exemplified by the amoebae Acanthamoeba and Naegleria [42,67] and the flagellate Oicomonas termo (possibly a species of Spumella) [68], can be cultured on dissolved organic matter, and many flagellates are reported to be osmotrophic [39], but evidence for the occurrence of osmotrophy in nature is circumstantial. Some flagellates rarely contain food vacuoles, and it has been suggested that uptake of dissolved organic matter is the only route of nutrient uptake in some small forms. Alternatively it may be used to supplement a particulate diet [69]. It has been argued, though, that osmotrophy plays only a minor role in nature. The low surface to volume ratio, of protozoa makes them bad competitors for dissolved nutrients compared to bacteria [46,70]. However, the concentrations of dissolved organic matter can be high locally, e.g. in hotspots, and this may allow growth of small osmotrophic flagellates. This is in accordance with the observation that small flagellates are able to increase in numbers just as fast as bacteria under favourable conditions (see below). while others are not eaten or even toxic to the a m o e b a e [6,72-74]. Singh [72] devised a method for evaluating the edibility of different bacteria. He arranged small pieces of glass tubes on agar plates so that they delimited small 'tracks' radiating from the centre of the agar plate. Each of the 'tracks' was streaked with bacteria and an inoculum of amoebae was placed in the centre of the plate. The amoebae in the centre moved in all directions and when they reached a suitable bacterial food supply they started eating the bacteria, multiplied and moved out along the bacterial streak. When they, on the other hand, reached a non-edible bacterial food, they either died or encysted and left the bacteria uneaten. Using this method Singh [6,72,73] tested the suitability of a large number of bacterial strains as food for two unidentified species of amoebae. The bacteria could be classified into three groups: some were readily eaten, some were partly eaten and allowed amoebal activity for a few days, after which the amoebae encysted or died, and some were completely avoided [73]. The inedibility of bacteria seems to some extent to be related to pigment production. Bacteria producing red, violet, blue, green or fluorescent pigments were observed to be inedible for two unidentified species of a m o e b a e [73,75] and to Leptornyxa reticulata [76]. The extracted pigments of strains of Pseudomonas pyocyanea, Chrornobacteriurn prodigiosum and C. l,iolaceum were found to be toxic to the two amoebal strains, the ciliate Colpoda steinii and the flagellate Cercomonas crassicauda. Groscop and Brent [77] found three out of 24 strains of pigmented bacteria to have a direct toxic effect on five strains of soil amoebae. Still, many colourless bacteria also proved to be inedible or unsuitable as food, so there must be other factors than pigment production that makes bacteria inedible or unsuitable as food for protozoa. Several workers have found Gramnegative bacteria to be a better food source than Gram-positive bacteria [78-80], but this was not confirmed by Severtzova [81] and Singh [43] who were unable to find any correlation between Gram-staining and edibility. 328 supported growth of some of the amoebae. Contrary to this the yeasts seemed to be suitable as food for the amoebae. All 19 species of yeasts were eaten by at least two of the amoebae and all four species of amoebae grew well on several of the yeasts. Nero et al. [85] found Acanthamoeba castellanii to grow well on the yeast Torulopsis famata. These results indicate that yeasts can be a suitable food source for bacterivorous amoebae, whereas mycelial fungi rarely will support growth of bacterivorous protozoa. Old [86] discovered that a giant amoebae, later identified as Arachnula impatiens [87], was able to feed on conidial spores of the plant pathogenic ascomycete Cochliobolus satit,us. These spores posses a melanized outer layer and are generally very resistant to biodegradation [88,89]. A. impatiens has also been found to feed on fungal mycelium in a similar manner [90]. After the discovery of this specialised mycophagous amoebae, several workers started to look for mycophagous amoebae by enrichment techniques instead of looking for them in old laboratory cultures grown on bacteria. Now representatives of several genera have been shown to feed on fungi. These include the three genera of giant amoebae: Arachnula [87], Theratromyxa and Vampyrella [91]. Arachnula impatiens invade fungal conidia by removing a disc-shaped portion of the conidial wall, after which the protoplasm is digested [89,92]. It is able to feed on fungal mycelium in a similar manner [90]. Some representatives of the genera Theratrornyxa and Vampyrella also feed on fungal spores by perforating the cell wall, but these organisms ingest the spores and form special digestive cysts [91]. Other examples of fungal eating amoebae are: Derrnamoebq (including Thecamoeba granifera ssp. minor [93]), Gephyramoeba, Mayorella and Saccamoeba [16], Rhizamoeba (including Ripidomyxa australiensis [94]), Deuteramoeba (including Trichamoeba mycophaga [95], which is the same species designated ('ashia mycophaga by Pussard et al. [96]), Hartmanella and Acanthamoeba [97]. Thus, the ability to feed on fungi seems to exist in many different and not closely related genera of soil amoebae. Downloaded from https://academic.oup.com/femsre/article/15/4/321/489287 by guest on 12 July 2022 Groscop and Brent [77] suggested that surface properties, such as the character of the bacterial coat, might be important. They noted that some of the inedible bacteria had a 'dry, waxy appearance'. Recently Gurijala and Alexander [82] observed that of several bacterial strains tested, those surviving protozoan predation in highest numbers had highly hydrophobic cell surfaces. The possibility that hydrophobicity of the cell surface may influence the ease with which cells are phagocytized by protozoa was also indicated by the observation that a species of Acanthamoeba ingested bydrophobic fungal spores less readily than hydrophilic [83]. Food preferences of bacterivorous soil flagellates have rarely been examined. Singh [6] found that the food preferences of the flagellate Cercomonas crassicauda resembled the food preferences of the two amoebae, but it was able to feed on a larger number of bacterial strains. Hardin [68] found Oicomonas termo (possibly a species of Spumella) to grow well on a large number of bacteria. Mitchell et al. [84] studied how attractive seven bacterial strains were for the flagellate Bodo saltans. It was allowed to migrate into either of two suspensions of different bacteria in T-shaped glass tubes. After 90 min the number of flagellates in each suspension was counted. Significant differences in numbers of flagellates present in suspensions of different bacteria were found. Differential protozoan feeding on bacteria in soil has rarely been examined. Singh [72] inoculated previously sterilized soil with amoebae and two species of bacteria of which one had previously been found edible in pure culture whereas the other had been found inedible. The amoebae grazed the edible bacteria but not the inedible. Consequently, the results of this experiment were in agreement with the results of the culture experiments on selective feeding. B. Mycophagy. Most of the earlier studies on mycophagy were carried out on cultures of bacterivorous protozoa. Heal [83] tested the edibility of 16 species of mycelial fungi and 19 species of yeasts by four species of amoebae. None of the amoebae were able to grow on mycelium of any of the fungi, but spores from two of the fungi 329 C. Feeding on eukaryotic organisms other than fungi. It is an established fact that several species of ciliates prey on other protozoa. According to Foissner [18] other protozoa have been found in the food vacuoles of approximately 50% of the 250 species of ciliates reported from soil. Species of testate amoebae have also been found to feed on other protozoa [18,98]. Many of the larger naked amoebae also seem capable of ingesting other protozoa. Thus Leptomyxa reticulata [102] and a mycophagous species of Mayorella [16] have been seen to ingest small amoebae. The mycophagous giant amoeba Arachnula impatiens sometimes ingest flagellates and is even able to feed on nematodes [87,92]. The giant amoeba Theratromyxa weberi engulfs nematodes and forms digestive cysts [103-105]. Another giant amoeba Vampyrella t,orax was found to contain small diatoms and flagellates in its digestive cysts, and though it has not been revealed which role these organisms play in the nutrition of the amoeba, it grew badly on bacteria and fungal conidia [91]. D. Detritit,ory. Many of the testate amoebae in soil can feed on humus particles [106]. Even though Tracey [107] found three species of naked amoebae to contain cellulase and chitinase, it is not generally believed that naked amoebae or flagellates are able to feed on resistant plant material or humus. Enumeration of soil protozoa, protozoan numbers and biomass Ecological research will often be based on information about numbers and biomass of the populations studied, and it is therefore unfortunate that heterotrophic flagellates and naked amoebae in soil are extremely difficult to enumerate precisely. It is even more difficult to say anything precisely about the ratio of encysted versus active individuals. Enumeration of soil protozoa: the MPN method The technique most commonly used for enumerating heterotrophic flagellates and naked amoebae in soil is the so-called most probable number (MPN) method originally developed by Cutler [108], and later modified by Singh [109], and by Darbyshire [110] and Darbyshire et al. [111]. The latter introduced the multi-diluter and the microtiter plate in the counting procedure. This modification is often used today. The principal idea of the method is to set up gradually more diluted series of soil suspensions in a suitable growth medium and then inspect the dilutions several times during the first month, to find out how much the soil has to be diluted before no protozoan growth can be observed. In this way an estimate of the total protozoan number (active + encysted) is obtained. It has often been assumed that direct microscopic examination of soil will yield a unreliable picture of the populations of naked amoebae, heterotrophic flagellates, and ciliates because their numbers are relatively small and because they cannot readily be separated from the soil particles [112]. On the other hand, it is generally accepted that the testate amoebae, which grow only slowly, are enumerated better by more direct methods, e.g. the membrane filter technique (see [113]). Unfortunately the MPN method suffers from serious shortcomings. The method will underestimate total protozoan numbers if organisms are killed during the set up of the cultures [114], or if they are not able to grow on the food offered. Ronn and Ekelund [115] showed that the number of protozoa as estimated by the most probable number method in some situations was more than 10 times higher when Enterobacter was used as medium than when an organic growth medium (tryptic soy broth 3 g/I) was used. Caron et al. Downloaded from https://academic.oup.com/femsre/article/15/4/321/489287 by guest on 12 July 2022 Only few species of amoebae are obligate mycophagous [98], e.g. Dermamoeba minor ( = Thecamoeba granifera ssp. minor), which feed on both hyphae and spores by perforating the cell wall and absorbing the cytoplasm [93]. A mycophagous flagellate isolated from soil, amended with conidia of the nematopbagous fungus Drechmeria coniospora, is apparently the first report of an obligate mycophagous flagellate [99]. There are also examples of obligate mycophagous ciliates [100,101]. 330 [116], working in aquatic systems, found that the dilution culture methods yielded at most 5% of the numbers from direct counts. Whether the statistical conditions for carrying out the culture methods, e.g. random distribution of organisms in the soil suspension, are fulfilled is also questionable [117,118] pected to harbour many active protozoa or so dry that protozoan activity could be regarded impossible. As Foissner [18] has pointed out, even a slight overestimation of the number of active protozoa compared to the total number will lead to a dramatic overestimation of the fraction of active organisms if the active fraction is small, which seems to be the case in many situations. The direct counting rnethod The number of actively feeding protozoa will be of more interest than the total protozoan number in most studies of decomposer populations. When the MPN method has been used to enumerate soil protozoa, it has often been combined with the ' H C I method' originally described by Cutler [108] in order to determine the number of active organisms. The idea of the method is to prepare two sets of dilutions, one of which is treated with 2% HCI for 16 h and afterwards neutralized with base. HC1 is supposed to kill all active protozoa, but not the encysted ones. The number of active individuals can be found as the difference between numbers in the two treatments. The method has been and is still frequently used (e.g. [9,112,119,120]). However, the HCI method has been heavily criticized. Bodenheimer and Reich [121] found that nearly all cysts of the common soil flagellates Heterornita globosa and Cercornonas spp. were killed by the HC1 treatment even at concentrations below 2%; all cysts of the amoeba Mayorella palestinensis were killed by the treatment at 22°C, but not at 9.5°C. Pussard and Delay [122] also found that cysts of some soil amoebae were killed by the treatment, and Berthold and Foissnet [118] observed the same phenomenon for soil ciliates. Ekelund and Ronn (in preparation) tested a gentle version of Curler's HCI method on different Danish agricultural soils. The pH was lowered to about 3 for 1 h, since preliminary liquid culture experiments had shown that this was just enough to kill all active protozoa. On average, 45% percent of the protozoa in the soils were killed by this treatment, the number being almost the same whether the soils were humid and ex- Lately a very simple technique has been proposed for enumerating soil protozoa: a portion of the soil is suspended in water or soil extract and examined under the microscope (for details see [123]). Foissner [18,124] and Liiftenegger et al. [123], using recovery experiments, showed that the efficiency of direct counting is high for testate amoebae and for actively swimming ciliates, but very low for naked amoebae. Foissner [18,21] has claimed that the direct counting method is useful for estimating numbers of active flagellates, too. In the present authors' opinion, however, the efficiency of direct counting for estimating numbers of active heterotrophic flagellates in soil is questionable. When they inspected agricultural soil directly they saw very few active flagellates, nearly all of them being actively swimming Spurnella-like forms, as opposed to the bulk of the flagellates seen when the culture methods are applied, these are mostly amoeboid flagellates moving along the substrate. The flagellate Foissner [18,124] used in his recovery experiment (Polytoma sp.) is a rather large, free-swimming form which is rarely observed in soil. The fact that Foissner [21] observed some flagellates in the litter layer of a spruce forest does not tell very much about efficiency of this method for enumerating active soil flagellates in general. Furthermore, an actively moving flagellate is not necessarily actively feeding: after food is depleted, flagellates may continue to swim actively for a considerable period spending very little energy, since their consumption of energy for maintenance is very small [46]. Finally, flagellates may en- and excyst very quickly. As mentioned above, Hughes and Smith Downloaded from https://academic.oup.com/femsre/article/15/4/321/489287 by guest on 12 July 2022 Actit'e z's. non actice soil protozoa: the "HCI method' 331 The number and biomass of protozoa in arable soil Numerous estimates of protozoan numbers in arable soil have been given using the methods described above. Different estimates will often be difficult to compare, since different counting methods have been used. In many situations, the counting methods are inadequately described, and in many papers no statistics have been applied. Estimates of parameters such as biomass and nitrogen mineralization are often calculated by combining results of countings with figures like those represented in Table 1. Such estimates are valuable food for thought but should be treated with caution since both protozoan numbers and conversion factors are encumbered with great uncertainty. Instead of giving a list encompassing most numbers from 1 to 16000000 per gram of soil, we will make some general remarks, based on the most probable number method as described by Darbyshire et al. [111]. Using this method, a differentiation between the different protozoan groups is difficult. Naked amoebae and hcterotrophic flagellates are usually equally numerous, while the number of testate amoebae and ciliates are about one order of magnitude lower; still, the biomass of the latter groups may be comparable or even larger than that of the first since the individuals are larger (Table 2; [18.126]). A large fraction of the soil protozoa will probably be in a non-active encysted state most of the time, but, since no reliable way to distinguish active from encysted protozoa seems to exist, we will desist from giving any estimates. The number of protozoa in soil may show very abrupt changes, caused by factors like hotspots [127], rhizosphere effects (R~nn and Ekelund, unpublished results), or rainfall [14]. When such factors are absent, however, the number of protozoa is fairly constant, probably mainly determined by the protectable pore space of the soil. The very. comprehensive work by Darbyshirc and Greaves [7] yielded numbers between 7000 and 70000 protozoa (amoebae and flagellates) per gram of soil in an unplanted soil kept at a constant moisture content of 35%, corresponding to 75% of water holding capacity. The numbers in a soil kept at 12% moisture content were in the same order of magnitude. In a pot experiment, in which the soil was allowed to dry as much as possible, Clarholm [64] found about 10000 amoebae and fewer flagellates per gram of soil. In a field study, where protozoan numbers were stud- Downloaded from https://academic.oup.com/femsre/article/15/4/321/489287 by guest on 12 July 2022 [65] showed that the flagellate Heteromita globosa was able to ex- or encyst in about 1 h, and anyone who studied flagellates under the microscope knows that they can round off and become inactive in a few minutes under the coverslip. Consequently, a soil protozoan may in theory switch from active to inactive several times between sampling and examination. While the direct counting method apparently yields reliable results for activity of ciliates and testate amoebae, at present it seems as if no reliable way exists to distinguish between active and encysted stages of naked amoebae and heterotrophic flagellates in soil. It is generally assumed that a large fraction is encysted, and in liquid cultures most forms will encyst when food is depleted; however, some will remain actively swimming for a long period of time. Still, if active flagellates are not necessarily actively feeding, and if the organisms are able to en- and excyst in a very short period of time, the concept of activity is very difficult to handle. Total protozoan numbers (active + encysted) can be considered a potential for grazing. The actual activity is perhaps measured better in a more indirect way, by recording changes in the total protozoan number from repeated enumerations carried out at intervals of a few days or even hours. From time to time, new methods for enumeration of soil protozoan populations by direct microscopy have been proposed (e.g. [125,114]), but such methods have never been widely adopted. A good method for direct enumeration of soil protozoa would be a great improvement, since the dilution methods suffer from being tedious, imprecise, and probably underestimating the protozoan number. Still, as long as a good method for direct enumeration has not been developed and widely accepted, the culture methods are necessary. In our opinion, the most reasonable thing to do is to try to improve the culture media. 332 led in bulk soil in a barley field during the growing season, numbers were found to be in the range 40000 to 100000 [128]. The 'basic' number of protozoa in a soil as estimated by the MPN technique is probably between 10 000 and I00 000 individuals (active + encysted) per gram of soil. Under favourable conditions, e.g. shortly after rainfall or after supply of easily decomposable organic matter, the number of protozoa may increase drastically. Clarho[m [64] found that the number of amoebae increased from 10000 to 60000 in an unplanted soil after addition of wa- Tablc 1 Some important ecological properties and conversion factors regarding soil protozoa, compiled from different sources 2.8-4.6 5 33 4.2-5.3 2.2-2.7 24-72 58 4.5 7 q.5 11.5 No growth 30 7 g 12 Little growth 6.6 7.2 12.9 No growth [236] Minimum generation times for different heterotrophic micro flagellates in monoxenic liquid culture with Pseudomonas at 20°C [122] Monoxenic cultures of different amoebae grown on Klebsiella in soil at 280( ` [237] Hartmanella and Naegh'ria grown monoxenic at 37°(7, log-phase [237] Hartmanella and Naegleria grown monoxenic at 37 ° C, log-phase [26] Natural population of small soil amoebae and flagellates [119] Natural population of soil amoebae in soil cores [238] Minimum generation time for Spumella sp. in liquid culture with Pseudomonas grown at 250(` Same as above, but grown at 2(I°C Same as above, but grown at 15°( ` Same as above, but grown at 10"(" Same as above, but grown at 5"C [239] Minimum generation time for C>rcomonas sp. in liquid culture with Pseudomonas grown at at 25°C Same as above, but grown at 20°( ` Same as above, but grown at 15c~(" Same as above, but grown at I{)':C Same as above, but grown at 5o( ` [151] Cercomonas sp. in sterilized soil culture with Pseudomonas grown at 20°C and - 6 0 kPa, value derivcd from figure. Same as above, but grown at - 300 kPa Same as above, but grown at - 100(I kPa Same as above, but grown at 30f10kPa Bacteria consumed pr. cell division 150 1000 300-400 2000-4000 5 00()- 12 000 129-150 [236] Different heterotrophic microflagellates in monoxenic liquid culture with Pseudomonas at 20°C [240] Presumed value for soil flagellates [240] Presumed value for soil amoebae [12] Acanthamoeha polyphaga with Pseudomona,s paucimobilis in a soil microcosm at 25°C [238] ,g'pumella sp. grown in liquid culture with t~'eudomonas at 10-25°C Yield (protozoan carbon produced per bacterial carbon ingested) 11.4 [10] Acanthamoeba casterllanii grown in soil microcosms with f~s'eudomonas eepacia 0.25 0.22-O.57 0.19 0.34-0.43 [203] Based on results by [10] but including cryptic growth [I 2] Acunthamoeba polyphaga with l;~eudomona paucimobilis in a soil microcosm at 25°C [241] Acanthamoeba polyphaga with P,seueh,nonas Paucimobilis in batch and continuous culture at 26°C [236] Different heterotrophic micro flagellates in monoxenic liquid culture with Pseudomonas at 20°C Biovolume (ttm 3) 2O 190 5-80 14 523 5O 4/)0 3 000 74 644 [236] Differenl heterotrophic microflagellates in growth at 20~(?, mean = 85/am ~ [236] Diflerent heterotrophic microllagellates, starving, mean = 34 p,m 3 [197] Natural assemblage of soil flagelhUes grown soil microcosms at 250( ` [26] Average soil flagellate [26] Average soil amoeba [26] Average soil ciliate ~' [18] Average soil ciliate " Downloaded from https://academic.oup.com/femsre/article/15/4/321/489287 by guest on 12 July 2022 Generation {doubling) time (hours) 333 Table 1 (c~,ntinued) Specific gravity 1.0 1./)4 [242] [243] Paramecium b [229] Active protozoan biomass [244] Soil protozoa [10] Amoebae or nematodes [241] Acanthamoeba polyphaga C/N ratio 8 4 3 5 5 5 7 l0 [196] Presumed value for soil biomass Schnflrer, unpublished results in [13] Presumed value for soil biomass [232] Presumed typical value for soil bacteria [245] Presumed typical value for soil bacteria [245] Presumed typical value for soil flagellate [245] Presumed typical value for soil amoeba [232] Presumed typical value for soil protozoa [156] Presumed average value for fungi ~' The biovolume of the average soil ciliate of Foissner [18] was calculated on basis of values from Foisner (Table 6 in [18]) assuming that Foissners average ciliate has the shape of an ellipsoid, the volume of which is: 4/3 x ~vX abe, where a, h, and c, are the semi-axes. The values of Stout and Ileal [26] and Foissncr [18] are vein different. This is probably because the value of [26] is based on the most common soil ciliates (possibly mostly ('olpoda steinii) while Foissners [18] value is the average value of all forms, including large and rare species. ~' Derived from sedimentation experiments with Paramecium where (p-p,~) was found to be 40 Kg/m); assuming a density of the liquid of 1.0 this gives a specific gravity of 1.04. ter, while Griffiths et al. [129] showed that p o p u lations as large as 1601t0000 flagellates a n d a m o e b a e were built up in a hotspot a r o u n d dead barley roots ( H o r d e u m I'ulgare). In the rhizosphere c o n s i d e r a b l e n u m b e r s may occur, too. Such large p o p u l a t i o n s may be built up very fast (see below), a n d it seems as if small flagellates are able to multiply faster t h a n the a m o e b a e in such situations, indicated by field a n d microcosm data [14,129] and by i n f o r m a t i o n from culture e x p e r i m e n t s (Table 11. Stout a n d H e a l [26] calculated from data of C u t l e r et al. [3] that the s t a n d i n g crop of small a m o e b a a n d flagellates would have a g e n e r a t i o n time of a b o u t 1 - 3 days. Brussard et al. [130] m a d e a c o m p a r i s o n of p r o t o z o a n b i o m a s s per ha in different c r o p p i n g systems, based both on their own results a n d estimates c o m p i l e d from other sources. T h e estimates from the different studies vary considerably, T h e lowest is 1.4 kg C per ha while the highest is 290 kg C per ha ( T a b l e 2). This very large variation may be caused both by differences in p r o t o z o a n biomass and by different ap- proaches to the estimation, a n d it is unlikely that all the differences are caused by actual differences in biomass. It has b e e n suggested for planktonic m a r i n e ecosystems that the biomass in logarithmically equal size groups would be roughly equal [131]. If this model is valid for the soil ecosystem, t h e n the biomass of bacteria (approx. 0 . 2 - 2 txm), a n d ' n a n o p r o t o z o a ' (approx. 2 - 2 0 txm) should be in the same o r d e r of m a g n i t u d e . This is obviously not the case in most of the studies in T a b l e 2. This might indicate that protozoan biomass often is u n d e r e s t i m a t e d . T h e effect o f p h y s i c a l / c h e m i c a l protozoa factors on soil Soil protozoa and p H T e s t a t e a m o e b a e will often prefer a rather n a r r o w p H range, outside which they only occur in small individual n u m b e r s . Miiller [132] d e m o n strated that m a n y species of testate a m o e b a e are Downloaded from https://academic.oup.com/femsre/article/15/4/321/489287 by guest on 12 July 2022 Dry weight pg tam 3 0.212 0.2 1).2 (/.I 334 tion of soil protozoa, as estimated by the MPN method, were illustrated by the work of Gupta and Germida [120]. They examined the effect of 5 years' repeated application of S ° fertilizer (22 or 44 kg Agrisul R per ha per year), resulting in a pH decrease from 5.7 to 5.2 and 4.7, respectively. Compared to untreated soil, both sulfur treatments resulted in a significant reduction in the number of bacterivorous protozoa and mycophagous amoebae. Since there was a concomitant decline in microbial, including fungal, biomass, it was concluded that the decrease in protozoan numbers was caused by a decrease in the amount of protozoan food as a result of the lower pH, perhaps accelerated by an increased concentration of sulfate. Soil protozoa and temperature Within a broad temperature range, increasing temperature will usually favour metabolism and activity and decrease population doubling time [65,137,138]. For many species examined, the upper temperature limit seems to be about 30°C with death occurring at 35-40°C [26,65] (see also Table 2 Protoz~mn. bacterial, and, funsal biomass (ks C / h a ) in arable soils at different sites under different cropping systems all figures are from [13(I] Site of research Protozoan biomass Bacterial biomass AI A2 13 C DI D2 EI E2 FI F2 10.5 13.0 149.8 289.7 50.1 40.0 139.2 3().5 1.4 1.0 648.5 829.6 ~91.6 440.3 700.9 899.8 - Fungal biomass 30.8 47.{) 15tt.2 159.1 151[11.(1 2101.5 - Bacterial +fungal biomass 679.3 876.6 458.6 263.7 841.8 599.4 2201.9 3001.2 232.65 270.27 Note that these kinds of estimates of protozoan biomass may be encumbered with serious uncertainty due to various conversion factors and difficulties with the enumeration methods (see sect. 4): as an example the MPN method probably underestimate the number of testate amoebae considerably (see [126] for more information on numbers of testate amoebae in agricultural soils). A, The Netherlands, silt loam, (}-25 cm, spring/summer, winter wheat; 1, conventional management; 2, integrated management; B, Alberta, Canada, silt clay loam, 0-10 cm, s u m m e r / a u t u m n , barley; C, Alberta, Canada, silt loam, 0-1(1 cm, s u m m e r / a u t u m n . barley. D, Georgia, USA, sandy clay loam, (I-15 cm, winter/spring, grain/rye, 1~ conventional tillage; 2, no tillage E, Sweden, loam, 0-27 cm, barley: 1. no N-fertilizer: 2, 120 kg N-fertilizer per ha: F, Colorado, USA, Mollisol, 0-10 cm, summer, fallow/wheat; 1, stubble mulch: 2, no tillage. Downloaded from https://academic.oup.com/femsre/article/15/4/321/489287 by guest on 12 July 2022 abundant in mor but not in mull; an elaborate formal classification of communities of testate amoebae with different pH preferences have later been developed [133,134]. Foissner [18] compiled results in this field from several sources and presented an updated list of character species of mor and mull. Only little information on the pH tolerance of other terrestrial protozoa than testate amoebae is available. On the basis of distributional and experimental data, mostly on ciliates, Stout [135] suggested that most protozoa will grow over a relatively wide range of pH values but have different pH optima. No effects of pH values between 4.2 and 8.7 on the growth of the amoebae Leptomyxa reticulata could be detected [76]. Stout and Heal [26] concluded that pH values from about 3,5-4.5 to 9.5 are the typical range for the more widespread soil protozoa. H o m m a and Cook [136], who examined the feeding behaviour of the mycophagous giant vampyrellid amoebae in many different soils, showed that it peaked in soils with a pH of about 7,0 and that no feeding could be detected in soils at either pH 4.0 or 8.5. The effects of pH changes on a total popula- 335 Soil protozoa t,s. oxygen/carbon dioxide Since shortage of oxygen and carbon dioxide surplus are closely connected phenomena, related to breakdown of organic matter in soil, they will be treated together. However, oxygen shortage is probably the most serious problem, since oxygen is only slightly soluble in water and diffuses very slowly; consequently, anaerobic microsites will exist temporarily even in well-aerated soils [27]. Carbon dioxide readily dissolves in the soil water, and may have an impact on soil p H but this is probably of minor importance. Since anaerobic conditions often occur where food is available, circumstantial evidence suggests that many soil protozoa can tolerate anaerobic or microaerophilic conditions. Sandon [139] suggested on the basis of the behaviour of organisms in culture that some soil protozoa tolerated or even preferred partially anaerobic conditions, but none of the attempts he made to obtain anaerobic cultures were a success. Another indication of tolerance of low oxygen levels among soil protozoa is that many of the forms reported from soil are common in sewage plants, in which oxygen tension is often low, e.g. Cercomonas and Mastigamoeba [37]. Moreover, the actual occurrence of Mastigamoeba and other m e m b e r s of the group ' A r c h e z o a ' in soil [21,140,141] clearly demonstrates the existence of anaerobic flagellates in soil. Recently, large protozoan numbers have been reported in connection with breakdown of organic matter in soil under very low oxygen tension [127,129]; and Ekelund and R~nn (unpublished results) showed that the growth of protozoa in soil, estimated by the MPN method, was virtually the same at oxygen tensions at 21, 14, and 7%, but that no growth occurred in an environment totally free of oxygen. The last finding is in accordance with Neff et al. [67], who showed that no growth of Acantharnoeba sp. occurred when oxygen was totally absent. Soil protozoa: soil water, texture and structure Two factors of special importance for the soil protozoa are the architecture of the habitable soil pore network, which is determined by soil texture and structure, and the soil moisture. The interaction of these factors establishes the basic environmental conditions for the soil protozoa. Most of the time, the aggregate structure of the soil will effectively restrict movement and only if the soil moisture is sufficiently high, movement of protozoa from one aggregate to another will be possible. Therefore, the protozoan fauna from different aggregates in the same soil may differ greatly [142-144]. The part of the soil pore network that is available to a particular species of protozoa will be restricted at the lower range by the size of the smallest pore necks the protozoa can penetrate. Furthermore, since protozoa (like their bacterial prey) are aquatic organisms and therefore restricted to the capillary water in pores between and within the aggregates in the soil, an upper limit for activity is determined by the largest water-filled pore necks. Downloaded from https://academic.oup.com/femsre/article/15/4/321/489287 by guest on 12 July 2022 Table 1). However, some amoebae, among others pathogenic species of Acanthamoeba and Naegleria, will grow at temperatures higher than 37°C. Cysts can survive much higher temperatures. The common soil flagellate Heteromita globosa has been shown to be active down to 1.5°C [65]. Stout and Heal [26] stated that no simple relationship between t e m p e r a t u r e and protozoan activity in soil has been shown; this statement is probably still valid. There are some reports of special adaptations to low t e m p e r a t u r e among soil protozoa. Hughes and Smith [65] showed that Heteromita globosa was the dominant species of Antarctic fellfields. Although Heteromita is pre-adapted to the harsh environment by having a low optimum temperature, the antarctic strain showed further adaptations (higher growth rate and larger percent excystment at low temperature), c o m p a r e d to an Aberdeenshire strain. Moreover, en- and excystment occurred in 1-2 h, thereby enabling it to make the most of the short periods with convenient temperatures. Rogerson and Berger [60] likewise reported that protozoa in a garden soil were able to en- and excyst quickly and in this way be active for short periods on many occasions throughout the winter. 33~ The diameter of the largest water-filled pore necks can be estimated from the water content of the soil, using the retention curve of the soil and the formula: d : 3000/10 p~ The ecological role of the soil protozoa Following this section on the role of protozoa in the food web and in mineralization processes, a special section is devoted to the role of protozoa in arable soil with special emphasis on root zone processes, since this subject has received special attention in relation to soil cultivation. The protozoa in the detrital food web The soil ecosystems are largely based on energy and nutrients from dead organic matter, which is utilized by the primary decomposers, i.e. bacteria and fungi; the primaD, decomposers are grazed by microfauna, protozoa and nematodes. While substantial information is available on the quantitative effects of protozoa on bacteria and to a lesser extent on the fungi in soil, very little exact information is available about which factors are important regulators of soil protozoa. Although an array of organisms are known to attack soil protozoa (see below), at present very little is known about the quantitative importance of different factors for regulation of the soil protozoan population. Several suggestions tor food web structures modelling the soil ecosystem have been proposed (e.g. [156,157]). In these models, the microfauna serves as food for higher wophic levels, for example predacious nematodes and microarthropods. The flagellates and the naked amoebae play a crucial role in these food webs. They are very important consumers of bacteria, and are eaten Downloaded from https://academic.oup.com/femsre/article/15/4/321/489287 by guest on 12 July 2022 where d (/zm) represents the largest waterfilled pore necks [145,146]. If the water content of the soil is so low that the size of the largest water-filled pore necks is smaller than the smallest pore necks that protozoa can penetrate, no protozoan activity will be possible. The size of the smallest pore neck that can be penetrated by a specimen will of course vary from species to species. Alabouvette et al. [147] calculated on the basis of data from Band and Umeche [148] that protozoan activity will be halted in soils in which pores larger than 6 > m arc devoid of water. An often assumed average lower limit for pore necks available to soil protozoa is 3 # m [19,149]; however, some small flagellates, e.g. Cercomonas can probably penetrate smaller pores, since they have been found to be active in very dry soils [150,151], and some giant amoebae have been reported to penetrate pore necks as small as l # m [87]. Another important p a r a m e t e r for predicting soil protozoan activity is the size of the part of the habitable soil pore network, which is inaccessible to potential predators. We will refer to that part of the soil as the protected pore space for protozoa, adopting a term that Postma and Van Veen [19] used for that part of the soil in which soil bacteria are not grazed. Nematodes, which may be important predators on protozoa, are supposed to be restricted by pore necks smaller than 30 p,m (occasionally 20 p.m for the small forms), confining them to the space between the aggregates or coarse particles [152]. The very common soil ciliate Colpoda steinii is restricted by pore necks of the same size [153], and ciliates are probably also important predators on smaller protozoa. Thus, the protected pore space for (small) soil protozoa is the water-filled fraction of the pores with necks in the range of 3 - 3 0 p,m, corresponding to values of pF in the range of 2-3. For a given water content, the number of water-filled pores of a particular size will differ for soils with different texture or structure [9]. Consequently, the number of habitable and protected pores in two different soils will be different. In a typical sandy soil, most water will bc found in pores with necks larger than 30 # m [146], leaving only a restricted protcctcd porc space for the protozoa, while a larger protected pore space will be found in a loamy soil. On the other hand, if the soil texture is too fine. protozoan activity may also be restricted due to lack of available pores [9,154,155]. 337 7he effect of protozoan grazing on bacterial populations Regulation of the size of bacterial populations Different factors can reduce the size of natural bacterial populations in soil. Physical factors (drying-remoistening, freezing-thawing) are known to be important in some situations [158,159], and an array of biotic factors like bacteriophages [160,161], bacterial parasites [162], and grazing by protozoa are recognized. The quantitative importance of biotic factors other than grazing, which is discussed in detail below, is not known. Most studies on protozoan grazing on bacterial populations have been carried out in the laboratory. A special line of work deals with the effect of protozoa on the survival of bacteria entering the soil. It was observed that populations of plant pathogenic bacteria and N-fixing strains of Rhizohium were able to maintain higher numbers when introduced into sterile soil compared to nonsterile soil [163-166]. The reduction in bacterial numbers in non-sterile soil was found to be paralleled by an increase in the number of indigenous protozoa. The bacterial populations maintained high densities when protozoan activity was inhibited by the eukaryotic inhibitor actidione (cycloheximide) [164,165,167]. These experiments provide strong evidence that protozoa play a major role in the regulation of bacterial populations. In experiments in which both protozoa and bacteria are added to sterile soil, it is usually found that numbers of bacteria are considerably reduced by predation compared to situations in which bacteria are added alone. However, they are not totally eliminated [4,72,168,169]. A number of hypotheses has been advanced to explain the inability of protozoa to eliminate their bacterial prey [170]. Three of these will be discussed briefly: (i) The inability of protozoa to eliminate bacteria is often observed also in liquid cultures. Thus, Danso and Alexander [171] found that populations of Rhizobiurn meliloti, which were kept in a salt solution, rapidly declined when grazed by amoebae, but the decline stopped when bacterial densities had fallen to about 10~-107 cclls/ml. The reason for the survival of the bacteria was not that they were resistant to attack; when they were concentrated and once more incubated with amoebae, 98% were consumed. The following explanation of bacterial survival was proposed: protozoa will either dic or encyst, and thereby stop feeding on bacteria, when the density of bacteria falls to a level where the energy gained by predation is equal to or less than the energy needed to find and consume the remaining bacteria [164,171]. (ii) The balance between protozoan and bacterial populations may also be maintained in a dynamic equilibrium because of bacterial multiplication. Thus, Habte and Alexander [172] showed that the ciliate Tetrahymena pyriformis was able to reduce bacterial numbers to much lower levels when bacterial reproduction was blocked by chloramphenicol, both in liquid culture and in re-inoculated sterile soil. (iii) The spatial heterogeneity of soil probably serves to stabilize the p r e d a t o r - p r e y relationship, because bacteria will survive in microsites where protozoa arc unable to graze upon them. In an attempt to test this hypothesis, Sardeshpande et al. [168] compared the survival of two bacteria when grazed by the ciliates Colpoda sp. and Uroleptus sp. in liquid culture and in previously sterilized soil. Downloaded from https://academic.oup.com/femsre/article/15/4/321/489287 by guest on 12 July 2022 by omnivorous and predacious nematodes. Small forms may also be eaten by some of the larger amoebae and the ciliates. Their small size enables them to utilize bacterial food which is inaccessible to the nematodes [9], (see above). Consequently, they form an important link between bacteria and higher trophic levels, Still, although food web theories like these seem trustworthy, they are based on few experimental data, and other types of interactions may be of equally great importance. In relatively dry soils or inside small soil pores where larger organisms are precluded from coming, other factors may be responsible for reducing the number of small flagellates and naked amoebae e.g. parasitism (see below). 33s Eflk'ct o[" protozoa on composition of microbial communities The observation that protozoa do not eliminate their prey is mainly related to grazing of single species populations. Mallory et al. [175] demonstrated that Salmonella typhimurium could be reduced to very low levels in sewage when large numbers of Enterobacter agglomerans ceils were added. They suggested that E. agglomerans served as an alternative prey, and when large number of cells of this organism were present in the sewage the protozoa could continue feeding, even if the numbers of S. typhimurium were low. The continued grazing may ultimately result in the elimination of the bacteria present in low numbers. Ramirez and Alexander [176] studied survival of Rhizobium inoculated on legume roots, and likewise found that the addition of an alternative prey caused reductions in numbers of the test rhizobia. When two or more species of bacterial prey are present in an environment, and at least one of these is present in densities above the threshold value for predation and another grows at a rate less than the predation rate, it is possible that the latter will be eliminated by predation [1751. The selection pressure imposed on the bacteria by protozoan predation favours bacteria that are able to avoid being eliminated by the predators. Sinclair and Alexander [177] found that bacteria with fast growth rates survived protozoan predation in higher numbers than bacteria with slow growth rates. Grazing has also been seen to favour bacteria which are inedible to protozoa because of their shape or size. Gfide [178] found that flagellate grazing on bacteria in activated sludge resulted in increased abundance of "grazing-resistant' spiral-shaped and filamentous bacteria. Bianchi [179] observed that grazing by flagellates and ciliates allowed growth of star-like and filamentous bacteria in sea-water. Grazing on a Gram-negative rod-shaped bacterium by the ciliate Cvclidium sp. resulted in the appearance of up to 20 /,m long cells on which the ciliate could not feed [180]. The long cells had significant slower growth rate than the normal rod-shaped cells, and in cultures without the ciliate predator they were out-competed by the more efficient rod-shaped cells. The above-mentioned experiments indicate that protozoan predation acts as a selection factor. Apparently this selection factor can influence bacterial populations in opposite directions. The bacteria can either avoid elimination by growing very fast, thereby replacing cells lost by predation (r-selection) or they can grow slower, and instead avoid consumption by attaining large size (Kselection). Downloaded from https://academic.oup.com/femsre/article/15/4/321/489287 by guest on 12 July 2022 They found that the bacteria were not eliminated in liquid culture, and concluded that the soil particles did not play any role in protecting the bacteria from being eliminated by grazing. Closer inspection of their data reveals, however, that the bacterial populations were grazed to considerable lower levels in liquid culture (3.1-4.6 × 104 ceils/ ml) compared to soil (1.2-9.0 x 10 ~' cells/g dry weight soil). This indicates that the bacteria were actually protected by the physical structure of the soil in the experiments by Sardeshpande et al. [168]. Furthermore, these experiments were carried out with introduced bacteria, which are not protected to the same extent as the indigenous bacteria. More recent studies have further stressed the importance of physical protection from grazing. Survival of introduced rhizobia was seen to be higher in soil amended with 10% bentonite clay than in unamended soil, probably as a result of the formation of microniches in which bacteria could not be reached by the protozoa [173]. Postma et al. [174] introduced bacteria to sterilized soil with or without the flagellate Bode) saltans, and enumerated the surviving cells in different soil fractions. In this way, free organisms and organisms associated with particles or aggregates were separated. The percentage of particle-associated bacteria was higher in the presence of the flagellate predator, indicating that the surviving bacteria were present in small pores in the aggregates or associated with the surface of soil particles. 339 Effect of protozoa on fungal populations The mycophagous amoebae have mainly been studied because of their potential regulation of plant pathogenic fungi. Populations of both mycophagous and total amoebae were found to be higher in soils sup- pressive to the take-all disease of wheat (caused by the fungus Gaeumannomyces graminis) than in non-suppressive soils [ 189,190]. Numbers of mycophagous protozoa have been seen to increase significantly upon addition of fungi to soils [99,147,191]. This might have a detrimental effect if mycorrhizal fungi are added to enhance root colonization or if fungi are inoculated as biological control of plant diseases. Chakraborty et al. [192] showed that mycophagous amoebae reduced the colonization of pine roots by the ectomycorrhizal fungus Rhizopogon luteolus, when added to soil at the samc time as the fungus. Protozoa may also affect fungal populations in a more indirect way via a stimulation of bacterial activity. Thus, the antagonistic effect of the bacterium Pseudomonas fluorescens on the fungus Fusarium oxysporum, which is probably exerted via production of siderophores, was observed to be enhanced by presence of Acanthamoeba castellanii in soil microcosms [193]. Lffect of protozoan grazing on decomposition and mineralization One of the aspects of protozoan ecology which has received most attention in recent years is the influence of amoebae and flagellates on mineralization and nutrient cycling in the soil. Mineralization, that is production of inorganic end-products (e.g. CO 2 and N H ] ) , is the final outcome of decomposition of organic material. Protozoan grazing is generally believed to stimulate the decomposition rate [70]. As naked amoebae and flagellates are not believed to consume particulate dead organic matter (see above), the influence of these organisms on decomposition rate must be exerted via an effect on the primary decomposers - bacteria and fungi. An example of a study where both decomposition rate and carbon mineralization were assessed at the same time is referred by Fenchel and Harrison [194]. Decomposition rate of 14Clabelled barley hay, measured as the rate of liberation of 14CO2, was rapid and nearly constant in seawater microcosms inoculated with bacteria and a natural assemblage of protozoa, while micro- Downloaded from https://academic.oup.com/femsre/article/15/4/321/489287 by guest on 12 July 2022 Stimulation of bacterial acti~'ity Presence of protozoa has been shown to stimulate a number of bacterial processes, e.g. nitrogen fixation [181-183], CO 2 evolution [9,184], nitrification [185] and siderophore production [186]. Pussard and Rouelle [187] showed that grazing by amoebae stimulated bacterial production. Based on countings of cell numbers with short time intervals, it was found that the total production of populations of Klebsiella aerogenes in previously sterilized soil aggregates was higher when the bacteria were grazed by the amoeba Acanthamoeba castellanii. Three different kinds of hypotheses have been proposed to explain the stimulatory influence of grazing on the bacterial activity and production: (i) Predation might selectively favour rapidly growing bacteria. (ii) Protozoa may excrete some organic growth factors that stimulate bacterial activity [181,182]. Hervey and Greaves [181] observed enhanced N-fixation by Azotobacter chroococcum in the presence of ciliates as well as heat-killed ciliate suspensions. (iii) When bacteria are grazed their populations are kept at a lower level and this prevents them from being limited by density-dependent factors, for example lack of nutrients, crowding, or excretion products. An ungrazed population is often controlled by some limiting resource, which causes the individual cells to grow very slowly [46]. Grazing releases nutrients immobilized in inactive microbial biomass, and this enables the remaining population to grow faster and maintain higher levels of activity [46,188]. Protozoa may also affect bacterial activity in a more indirect way, by transporting bacteria on their surface and allowing them to colonize new substrate [173]. 34() cosms only inoculated with bacteria had a lower and declining rate. Systems inoculated with bacteria and one choanoflagellate species took an intermediate position. Effect on C-mineralization It has often been observed that cumulative CO 2 evolution is higher in microcosms when protozoa are present than when they are absent [5,10,184,195,201,202]. It is generally agreed that the contribution from the protozoa to total respiration is of minor importance [203], and the enhanced CO 2 evolution must consequently be due to an effect on bacterial activity. The effect of protozoan grazing on the bacteria may influence carbon mineralization in two opposite directions. The stimulatory influence on bacterial activity will tend to increase total respiration, while the reduction in bacterial numbers will tend to decrease it. High grazing pressure may result in 'overgrazing', which will reduce the amount of CO 2 produced. De Telegdy-Kovats [184] found the total respiration to be higher when both bacteria and protozoa were added to sand cultures than when only bacteria were added, but repeated additions of protozoa, leading to higher numbers of protozoa, Downloaded from https://academic.oup.com/femsre/article/15/4/321/489287 by guest on 12 July 2022 The use of soil microcosms in studies of mineralization Since it is extremely difficult to obtain unequivocal information on the role of protozoan populations in mineralization and nutrient cycling from field studies, a more reductionistic approach is necessary, and much valuable information has come from soil microcosm studies. A common approach is to compare the parameter of interest in microcosms containing previously sterilized soil re-inoculated either with bacteria alone or with bacteria and protozoa. A microcosm experiment is consequently carried out in three steps: sterilization, re-inoculation and sampling. In the extensive studies carried out at Fort Collins by Coleman and co-workers, soil was sterilized by fumigation with propylene oxide [8,9,195]. When this method is used, substantial amounts of propylene glycol, which may serve as substrate for bacterial growth, are left in the soil. Other workers sterilized soil by autoclaving [196,197] or g a m m a irradiation [153,198]. Sterilization affects physical, chemical and biological properties of the soil (see e.g. [199,200]), and this will influence growth of the inoculated organisms. No matter which method used, killed microbial biomass and partly destabilized soil organic matter constitute easily accessible sources of carbon and nutrients for the inoculated organisms. Therefore strong bacterial growth will usually occur in the first days after re-inoculation of sterilized soils. However, inhibiting effects of autoclaving and irradiation on growth of bacteria and ciliates in previously sterilized organic forest soil have also been observed [200]. This inhibition was attributed to toxic organic substances produced during sterilization, and this effect is probably of less importance in mineral soils. Inoculation of the microcosms can be done with pure cultures of a limited number of known organisms. In this case the microcosms are said to be 'gnotobiotic'. Such gnotobiotic microcosms have been widely applied, for example by the workers at Fort Collins (e.g. [9,10]). The advantage of working with these biologically well-defined systems is that the interactions between different organismal groups are relatively simple and the results are therefore more easily interpreted. Still, the bacterial and protozoan communities in the natural soil environment consist of a number of species, and attempts have been made to include this complexity in microcosm studies. In stead of inoculating with only one or a few species of bacteria, a natural assemblage of soil bacteria can be prepared by filtering a soil suspension [198]. Frey et al. [197] inoculated microcosms with soil suspensions filtered through different pore sizes, and in this way they obtained microcosms with different portions of the protozoan fauna. The major part of the organisms may be killed during inoculation. Of the three protozoan groups, flagellates, amoebae and ciliates, the flagellates showed the greatest reduction in numbers upon addition to soil, whereas ciliates that were least affected still showed more than 70% reduction in numbers upon inoculation [197]. 341 PRB = (RB × Y B ) / ( 1 -- YB) in which RB iS the total respiration during the experiment and Y~ is the bacterial yield. The yield of bacteria was estimated in a separate experiment using ~4C-labelled glucose to be 11.6 [10]. The yield of the grazers (Yp) could now be estimated by the formula: Yp = B e / ( PRu - BB) or by insertion of the formula for PRu: Yp = B p ( 1 - Yh)/(RBYB-- BB(1 -- YB)) in which Bp is the amount of carbon in the biomass of the grazers and B B is the carbon present in the bacterial biomass. From this, CoOteaux et al. [203] estimated the yields of amoebae and nematodes in the experiments by Coleman et al. [10] to be approximately 0.25 and 0.02, respectively, On the basis of these lower yields of the grazers they were able to calculate the total amount of bacterial biomass consumed by the grazers. The total amount of carbon assimilated by the bacteria could then be estimated, and it was found to be considerably higher (1466%) in the grazed microcosms compared to the ungrazed. The 'extra' carbon which was assimilated by grazed bacteria was believed to be present as metabolites and perhaps organic material resistant to degradation. These calculations indicate that grazing actually did increase carbon use and bacterial production in the experiments by Coleman et al. [10]. The inclusion of cryptic growth in the calculations by CoOteaux et al. [203] is reasonable. Estimation of bacterial production simply as differences between bacterial counts with given time intervals wil! probably not result in reliable estimates. The calculations by Cofiteaux and coworkers show that estimates of protozoan biomasses can be used for calculating the actual amount of bacterial production. Sinclair et al. [205] examined the effect of amoebal grazing on N- and C-mineralization of the bacterium Pseudomonas paucimobilis in liquid culture. When nitrogen was limiting, grazing caused a higher bacterial production. There was no effect on the CO 2 evolution or the total amount of carbon used, and the higher bacterial production was therefore attributed to a more Downloaded from https://academic.oup.com/femsre/article/15/4/321/489287 by guest on 12 July 2022 had a negative influence on the total amount of CO 2 produced. He concluded that a biological equilibrium between the number of bacteria and protozoa exists, and that disturbance of this equilibrium in either direction will result in reduced CO 2 evolution. The interpretation of microcosm experiments is not straightforward, because it is difficult to assess the flow of carbon through the different compartments of organic carbon. Higher cumulative CO 2 evolution does not necessarily imply that the total amount of carbon assimilated by the decomposers is higher as well. Coleman et al. [10] studied the effect of grazing by nematodes a n d / o r amoebae on the flows of carbon in gnotobiotic soil microcosms. The total amount of CO 2 produced was higher in the grazed than in the ungrazed microcosms, but the sum of biomass-C, based on estimates of numbers of organisms, and CO2-C was not affected by grazing. Therefore, the authors concluded that grazing did not increase total assimilation of carbon, but merely caused a different allocation of carbon between biomass and CO 2. The yields of amoebae and nematodes, when feeding on bacteria, were found to be 0.4 and 0.04, respectively. These yields were estimated on the assumption that the number of bacteria ingested by the grazers could be estimated as the difference between the number of bacteria in ungrazed and grazed systems, i.e. they disregarded a possible higher growth of bacteria in the grazed systems (cryptic growth [204]). Cofiteaux et al. [203] re-examined the data presented by Coleman and co-workers and attempted to take cryptic growth into account. They assumed that only a minor part of the carbon assimilated by the bacteria was used for maintenance, in which case the ratio between total production and total respiration is equal to the ratio between the fraction of assimilated C going to production (yield) and the fraction going to respiration (1 - y i e l d ) , and the total bacterial production in the system (PR B) could be estimated by the formula: 342 efficient carbon use, probably due to higher availability of nitrogen. Predators and parasites on soil protozoa A number of organisms are known to be predators or parasites on soil protozoa. Among the metazoans, nematodes have been demonstrated several times to ingest protozoa [213]; this type of interaction was studied in more detail in a microcosm experiment [9]. Astigmatic mites have also been reported to ingest protozoa, mostly ciliates and testate amoebae [214]; protozoa seem to constitute a substantial part of the food taken Downloaded from https://academic.oup.com/femsre/article/15/4/321/489287 by guest on 12 July 2022 Effect on N-mineralization A number of studies has shown that grazing by protozoa enhances nitrogen mineralization, measured as ammonium production, both in liquid cultures [205,206] and in soil microcosms [195,207]. Contrary to carbon mineralization, in which the direct contribution from protozoan respiration is believed to be of little importance, the protozoa may directly excrete significant amounts of ammonium [208]. It has been assumed that when protozoa feed on bacteria with approximately the same C / N ratio as their own, they will incorporate one-third of the nitrogen into their biomass, excrete one-third as organic nitrogen and excrete one-third as ammonium [209]. Consequently, enhanced N-mineralization in systems with protozoa may be due to a stimulation of bacterial activity or to direct excretion of inorganic nitrogen by the protozoa. Since bacteria are efficient scavengers of inorganic nutrients they are not believed to excrete these to any great extent during active growth [210]. It is therefore most likely that most of the nitrogen which is mineralized passes through the grazers. The results of a microcosm study concerning the effect of amoebae and nematodes on Nmineralization illustrate the importance of direct excretion of N by grazers compared to increased bacterial activity [207]. The two different grazers affected the respiration and bacterial populations in a similar way, and if their effect on N-mineralization were solely due to an effect on bacterial activity they would be expected to have the same effect on N-mineralization. This was not the case, however: the amoebae always increased Nmineralization, whereas nematodes only did this when their populations declined. This was attributed to a difference in the life history of the two grazers, and the hypothesis was put forward that the nematodes changed from excreting N in organic form when bacterial food was plentiful to excreting N as ammonium when the bacterial food supply was exhausted and the nematode populations therefore declining. Effect on mineralization of other nutrients Mineralization of sulfur has rarely been examined. Gupta and Germida [155] found that mineralization of sulfur was enhanced in microcosms grazed by amoebae, and there was a significant negative correlation between sulfur in the microbial biomass and extractable SO4-. Mineralization of phosphorus has been shown to be enhanced in the presence of amoebae in soil microcosms [11,195], but the underlying mechanisms behind this are not fully understood. Johannes [188] found that regeneration of dissolved inorganic phosphate from organic detritus in aquatic microcosms proceeded faster when ciliates or flagellates were present than when bacteria were present alone. He attributed this to direct excretion from the protozoa. Barsdate et al. [211] also found protozoa to enhance phosphorus mineralization in aquatic microcosms, but only a minor part of this phosphorus passed through the protozoa. They therefore suggested that grazing changed the physiology of the bacteria, thereby promoting a more rapid phosphorus cycling. It is likely that mineralization of nitrogen in the presence of protozoa is primarily a result of direct excretion from protozoa, but this does not necessarily apply to mineralization of phosphorus. It has been proposed that organic P, which exists as esters (C-O-P), is mineralized by bacterial exo-enzymes to a larger extent than organic N, and that P-mineralization is controlled by a need for the element, whereas the mineralization of organic N, which is directly bonded to C, to a larger extent is driven by the need for energy [212]. 343 Protozoa in arable soil and their relation to plant growth Protozoa in arable soil have received special attention because of their potential significance for the cultivated plants. Their importance for the processes in the soil in close contact with the roots, the rhizosphere, has been the focus of several studies. Protozoa in the rhizosphere The rhizosphere is a highly favourable habitat for microorganisms [27], such as fungi and bacteria. Since these organisms are the major food components of soil protozoa, it is not surprising that the rhizosphere often harbours a higher number of protozoa than the surrounding soil. Darbyshire and Greaves [223] have given a short review of the bacteria and protozoa in the rhizosphere. The rhizosphere effect The term rhizosphere effect is used for the number of organisms in the rhizosphere proportional to the number in the bulk soil. Several estimates of the rhizosphere effect for protozoa in different situations and habitats have been given. Darbyshire [224] who mentions older works, attaches importance to the fact that few of the workers have applied statistical tests to their estimates. Rouatt et al. [225] were the first to use statistics in the analysis of the rhizosphcre effects which they found to be in the range of 2.4 to 3.4. Some of the more comprehensive recent work done on rhizosphere protozoa will be treated in more detail below. Reports of number of species in rhizosphere soil versus non-rhizosphere soil are somewhat contradictory, partly because different workers are using different definitions of rhizosphere and non-rhizosphere soil and different methods in separating them. Bicz6k [226] found a higher number of species and individuals in the rhizosphere of wheat than in unplanted soil, while Bamforth [227] found quantitative rhizosphere effects from 3 to 60 in sub-tropical forests in south-eastern Louisiana, USA, but a reduced species diversity; statistical tests were not applied in any of these works. Darbyshire and Greaves [7] grew the annual Sinapis alba and the two perennials, Trifolium repens and Loliurn perenne in pots at a constant moisture content of 35%, and examined the rhizosphere and the non-rhizosphere protozoa over a set period of time. They found that already from an early stage in the experiment the number of protozoa in the rhizosphere of Sinapis and Trifolium was significantly higher than in the unplanted soil. For both plants the largest rhizosphere effect was found during the early stages of flowering, when the highest number, 225 000 protozoa per gram of soil, was found in pots planted with Sinapis alba. There was no difference in the species composition between the rhizosphere and the unplanted soil, but in several situations species of the flagellate genera Heteromita and Cercomonas were significantly more abundant in the rhizosphere than in the unplanted soil. Although a rhizosphere effect was indicated at all four samplings during a 98-day period, it was only significant on day 71 in the case of Lolium perenne. However, members of the genera Heteromita and Cercomonas were significantly more abundant in rhizosphere soil on several occasions. On day 71 and 98, Lolium perenne was studied at two different moisture contents, 12% and 35%. A Downloaded from https://academic.oup.com/femsre/article/15/4/321/489287 by guest on 12 July 2022 up by the earthworm Eisenia foetida [215], and active protozoan cells seem to be digested by other earthworms, too [216,217]. Among the protists, ciliates, testate- and larger naked amoebae have been shown at several occasions to ingest other protozoa including small naked amoebae and flagellates (see above). The small flagellates of the genus Colpodella (Spiromonas) attack flagellates and ciliates and withdraw cytoplasm from the prey with a 'rostrum' [218,219]. In a number of cases, fungi parasitizing ciliates and testate amoebae have been demonstrated [18], and several zygomycetes capture naked amoebae and assimilate them [220,221]. Bacterial endoparasites on naked amoebae in soil have also been demonstrated [222]. 344 population was similar to that of the bacteria with a peak 2 days after rainfall. Griffiths [229] only found rhizosphere effects in some situations when working with Hordeum Bacteria g ~ t,ulgare, Pisum satil,um, Brassica rapifera and Lolium perenne. Naked amoebae 041 campestri~" Ef[ects of protozoa on plant growth ~02 O 2 4 6 8 Days 10 12 14 "; 16 Fig. 1. Changes in biomass (mg dry w e i g h l / g dry weight of soil) of bacteria and naked amoebae in pots after watering at day 0; before watering the pots were allowed to dry out, subsequent addition of water on day 6 and day 12 had no effect on the populations. Each pot contained 14 19-day-old wheat plants in 300 g soil al day 0. After Clarholm [64]. positive rhizosphere effect could only be observed at 35%. During the growth season of a species of Panicurn, Napolitano [228] found a rhizosphere effect larger than 1 in almost all of 17 samplings; in accordance with Darbyshire and Greaves [7] the effect declined after flowering. In a pot experiment with wheat plants (Triticum aestit,um), numbers of amoebae in dry soil were approximately l(t 4 per gram of soil, both in planted and unplanted soil (Fig. 1). Addition of water quickly induced bacterial growth, and the bacterial population reached a peak in l - 2 days. Subsequently, the number of amoebae increased, and 5 days after watering the number of amoebae had increased six-fold in the unplanted soil and 30-fold in the planted soil. Parallel to the increase in amoebal numbers, the number of bacteria was greatly reduced again. After 2 more days, the original number of amoebae was reduced two-fold and three-fold in unplanted and planted soil, respectively [64]. In a field experiment with barley (Hordeum culgare), Clarholm [14] observed a similar effect after rainfall on the ratio of a m o e b a e in planted vs. unplanted soil: the population of soil a m o e b a e peaked after 4 days, whereas the temporal variation of the flagellate Downloaded from https://academic.oup.com/femsre/article/15/4/321/489287 by guest on 12 July 2022 C Since protozoa play an important role in mineralization and have an increased abundance in the rhizosphere, they may play an important role in plant nutrition. The works cited below suggest that this is actually the case. Elliott et al. [230] used microcosms with propylene oxide-sterilized soil to study the effect of amoebae on nitrogen uptake by plants. The prairie grass Bouteloua gracilis was grown with the bacterium Pseudomonas cepacia, with or without the amoeba Acanthamoeba polyphaga. Both microorganisms were isolated from the rhizosphere of the grass. Glucose and three different levels of NH 3+ N were added to the microcosms. After 60 days, nitrogen contents in shoots were significantly higher in the presence of amoebae, while total plant uptake of nitrogen only was increased by the presence of amoebae at the medium N-level. A m o e b a e had no effect on plant yield. Clarholm [196] studied the effects on wheat (Triticum aestit,um) of inoculating autoclaved soil with a mixed population of soil bacteria either alone or in combination with a mixed population of soil protozoa. Glucose and ammonium nitrate were added to the microcosms twice a week. At harvest after 42 days, she found that presence of protozoa had resulted in plants with a significantly higher biomass and N-contents in shoots, while there was little variation between the different treatments for the roots. Kuikman and Van Veen [198] grew wheat (Triticum aesticum) in microcosms with gammairradiated soil, with bacteria, with or without a mixed population of soil protozoa. Neither additional carbon nor fertilizer were added. After 14 days the nitrogen concentration was higher in the shoots and a little lower in the roots in the treatments with protozoa, while other plant pa- 345 Clarholm ~ hypothesis Clarholm [13,209] has proposed a model to explain the interactions leading to mineralization of soil organic N and subsequent plant uptake. The model is developed from a model by Elliott (in [231]): Organic carbon is excreted by the plant into the surrounding soil as the root tip grows. The excreted carbon results in bacterial growth around the root. The growing bacteria mineralize and immobilize soil organic nitrogen in bacterial biomass nitrogen. The bacteria around the root will later be consumed by protozoa. When the protozoa excrete part of the bacterial nitrogen as ammonia in the vicinity of the root, the plant can take up the nitrogen. In a mathematical model, Robinson et al. [232] tested the hypothesis that root-derived carbon may lead to enhanced mineralization of soil organic nitrogen and subsequent plant uptake. Some of the key factors in their analysis were, among many others: the C / N ratio of soil organic matter and root-derived substances, the availability to bacteria of soil-derived substances compared to plant-derived substances, and presence or absence of protozoa. Not surprisingly, their conclusions were among others that in general it would be most beneficial for the plant if the C / N ratio of the root-derived organic matter was relatively high, and that grazers in the rhizosphere will have a critical influence on the ability of the plant to take up soil organic nitrogen. On the basis of the assumptions of the model, however, the root-induced mineral- ization of soil organic nitrogen could at most account for about 10% of the maximum plant uptake that has been measured. Still, many of the parameters included in the model arc based on few experimental data, and the paper gives no information on sensitivity of the model output to slight changes in parameter values. Moreover, the authors conclude that in some situations the effect of root-derived substances can be crucial for the plant uptake of nitrogen. Although Robinson and co-workers question the importance of the mechanism proposed by Clarholm for plant uptake of mineralized soil nitrogen, they do not question the importance of grazers for mineralization of soil nitrogen in general. In fact, their model shows that soil nitrogen will not be mineralized by bacteria alone unless the C / N ratio of the available soil organic matter is below 5, which is rarely the case in whole soil, and they concluded that "Nitrogen mineralization would always depend on the grazing of bacteria by predators" [232]. The last conclusion might seem to be inconsistent with the results of the microcosm experiments cited above [196,198,230] which show that although more nitrogen is mineralized in the presence of protozoa, a great amount is mineralized by bacteria alone. However, the source of nitrogen in the microcosm experiments was not only the soil organic matter. In two of the experiments glucose and inorganic nitrogen were added to the microcosms, and in all three experiments sterilization of the soil added easily decomposable, nitrogen-rich organic matter to the soil in the form of dead microbial biomass. Grazers may be more important for remineralization when pools of easily available organic nitrogen are not artificially increased. Still, an experiment in soil without any predators, but otherwise unaltered, will be very difficult if not impossible to carry out. Other interactions between protozoa and plants Positive effects of protozoa on plant growth can be caused by other factors than enhanced nitrogen mineralization. Some older papers are cited in [233]. Nikoljuk and Tapilskaja [234] showed that an amoeba isolated from the rhizo- Downloaded from https://academic.oup.com/femsre/article/15/4/321/489287 by guest on 12 July 2022 rameters were similar. On day 35 nitrogen concentration in both shoots and roots was higher in treatments with protozoa. Shoot biomass was elevated in the presence of protozoa, too, but root biomass was reduced, resulting in an unaltered total plant biomass in the presence of protozoa. Clarholm [14] calculated that bacterial-protozoan interactions contributed 10-17% of the nitrogen taken up by barley (Hordeum L~ulgare); the result being the same whether calculations were based on the extent of predation or the amount of carbon coming from root exudates. 346 Important literature Some of the references listed below are cited several times in the text above, and deserve a special mentioning. Sandon's [139] monographic work is still of great value, it contains a wealth of information regarding autecology. The extensive review of Stout and Heal [26] treats most relevant literature from the period 1927 to 1967. The more functional aspects of soil protozoa have been treated in many papers by the Fort Collins group in Colorado (Anderson, Elliott, Coleman, Cole, and Bryant among others) and by Clarholm; their papers can be found among the references below. Lately, Fenchel [46] published a very useful book on protozoan ecology, and the autecological work of Foissner, especially Foissner [18], is also of great value. Most of the naked a m o e b a e are covered in the excellent key by Page [32], and an extensive and updated account of flagellate taxonomy and morphology is given by Patterson and Larsen [39]. References 1 Russell, E.J. and Hutchinson, H.B. 1191)9) The effect of partial sterilization of soil on the production of plant fi~od, J. Agricult. Sci. 3, 111-114. 2 Martin, C H . and Lewin, K.R. (1914) Some notes on soil protozoa. Phil. Trans. Roy. Soc. London, Set. B 05, 77-94. 3 Cutler, D.W., Crump, L.M. and Sandon, H. (1~22) A quantitative investigation of the bacterial and protozoan population of the soil, with an account of the protozoan fauna. Phil. Trans. Roy. Soc., B. 211,317 350. 4 Cutler, D.W. (lt)23) The action of protozoa on bacteria when inoculated into sterile soil. Ann. Appl, Biol. 10, 137 141 5 Cutler, D.W. and Crump, L.M. 11929) Carbon dioxide production in sands and soils in the presence and absence of amoebae. Ann. Appl. Biol. 16, 472-482. 6 Singh, B.N. 11942) Selection of bacterial food by soil flagellates and amoebae. Ann. Appl. Biol. 29, 18-22. 7 Darbyshire, J.F. and Greaves, M.P. (1967) Protozoa and bacteria in the rhizosphere of Sinapis alba L., Trifolium r~7~ens L.. and Lolium perenne L. Can. J. Microbiol. 13, 11157-1 {_)68. 8 Anderson, T.R., Elliott, E.T., McClellan, J.F., Coleman, D.C., Cole, C.V. and Hunt, H.W. 11978) Trophic interactions in soils as they affect energy and nutrient dynamics. 111. Biotic interactions of bacteria, amoebae, and nematodes. Microb. Ecol. 4, 361-371. 9 Elliott, E.T., Anderson, R.V., Coleman, D.C. and Cole, C.V. (1980) Habitable pore space and microbial trophic interactions. Oikos 35, 327-335. 10 Coleman, D.C., Anderson. R.V., Cole, C.V., Elliott, E.T., Woods, L. and Campion, M.K. (1978) Trophic interactions in soils as they affect energy and nutrient dynamics. IV. Flows of metabolic and biomass carbon. Microb. EcoL 4, 373-38/). I 1 Cole, C.V., Elliott, E.T., Hunt, H.W. and Coleman, D.C. (1978) Trophic interactions in soils as they affect energy and nutrient dynamics. V. Phosphorus transformations. Microb. Ecol. 4, 381-387. 12 Bryant, R.J., Woods, L.E., Coleman, D.C., Fairbanks, B.C., McClellan, J.F. and Cole, C.V. (1982) Interactions of bacterial and amoebal populations in soil microcosms with fluctuating moisture content. Appl. Environ. Microbiol. 43, 747-752. 13 Clarholm, M. (1983) Dynamics of soil bacteria in relation to plants, protozoa and inorganic nitrogen. Ph. D. thesis, Swedish University of Agricultural Sciences, report no. 17, Uppsala. 14 Clarholm, M. (1989) Effects of plant-bacterial-amoebal interactions on plant uptake of nitrogen under field conditions. Biol. Fertil. Soils 8, 373-378. 15 Kuikman, P. (I990) Mineralization of nitrogen by protozoan activity in soil. P h . D . thesis, Landbouwuniversiteit, Wageningen. Downloaded from https://academic.oup.com/femsre/article/15/4/321/489287 by guest on 12 July 2022 sphere of lucerne (Medicago sativa) had a positive effect on young lucerne plants, caused by the production of the plant hormone heteroauxin. Gould et al. [235] demonstrated that the prairie grass Bouteloua gracilis produced more acid phosphatase when grown in the presence of Pseudomonas cepacia or in the presence of Pseudornonas cepacia and the amoeba Acanthamoeba polyphaga, than it did when grown alone. Plants with both amoebae and bacteria produced more phosphatase than plants with only bacteria, but the difference was not significant. Protozoa may also play a role in the regulation of plant pathogens (see above). Under some circumstances, the presence of protozoa in the rhizosphere is regarded detrimental because they prey on introduced bacteria. In a study where germinating seeds of leguminous crops were inoculated with rhizobia, Ramirez and Alexander [176] found root colonization to be enhanced when protozoan inhibitors were administered to the soil. 347 of strains in culture. In: The Biology of Free-living l:leterotrophic Flagellates (Patterson, D.J. and Larsen, J., Eds.), pp. 477-492. Systematics Association. Clarendon Press, Oxford. 34 Margulis, L., Corliss, J.O., Melkonian, M. and Chapman, D.J. (1990) Handbook of Protoctista. Jones and Bartlett Publishers, Boston, MA. 35 Pascher, A. (19131 Heft 2, Flagellatae 2. In: Die Siisswasser-flora Deutschlands, Osterreichs und der Schweiz (Pascher, A., Ed.), Gustav Fischer Verlag. Jena, 36 Lemmerman, E. (19141 Heft 1, Flagcllatae 1. In: Die Sfisswasser-flora Deutschlands, ()sterreichs und der Schweiz (Pascher, A., Ed.), Gustav Fischer Verlag, Jena. 37 Hiinel, K. (19791 Systematik und {Skologie dcr farblosen Flagellaten des Abwassers. Arch. Pmtistcnk. 12 I, 73-137. 38 Patterson, D.J. and Hedley, S. (1992) Free-living Freshwater Protozoa. Wolfe Publishing Ltd., London. 39 Patterson, D.J. and Larsen, J. (1991) The Biology of Free-living Hetcrotrnphic Flagellates. Systematics Association, Clarendon Press, Oxford. 411 Page, F.C. and Siemensma, F.J. (19911 Nackte Rhizopoden und Heliozoa, Protozoenfauna Band 2. Gustav Fischer Verlag, Stuttgart. 41 Page, F.C. (1987) The classification of 'naked' amoebae (Phylum Rhizopoda). Arch. Protistenkd. 133, 199-217. 42 Schuster, F.L. (19791 Small Amebas and Ameboflagellares. In: Biochemistry and Physiology of Protozoa, 2nd. edn. (Levandowsky, M. and Hutner, S.H., Eds.), Vol. 1, pp. 215-285. Academic Press. New York, NY. 43 Singh, B.N. (19751 Pathogenic and Non-pathogenic Amoebae. Macmillan, London and Basingstoke. 44 Lalitha, M.K., Anandi, V., Srivastava, A,, Thomas, K., Cherian. A.M. and Chandi, S.M. (19851 Isolation of Aeanthamoeba culhertsonii from a patient with meningitis. J. Clin. Microbiol. 21,666-667. 45 Reference omitted. 46 Fenchel, T. (19871 Ecology of Soil Protozoa. Science Technical, Madison, Wl. 47 Dyer, B.D. (1990) Phylum Zoomastigina Class Amebomastigota. In: Handbook of Protoctista (Margulis, L., Corliss, J.O., Melkonian, M. and Chapman, D.J., Eds.), pp. 186 190. Jones and Bartlett Publishers, Boston, MA. 48 Schuster, F.L. (19901 Phylum Rhizopoda. In: Handbook of Protoctista (Margulis, L., Corliss, J.O., Melkonian, M. and Chapman, D.J., Eds.), pp. 3-18. Jones and Bartlett Publishers, Boston, MA. 49 Mignot, J.-P. and Raikov, 1. (19921 Evidence for meiosis in the testate amoeba Arcella. J. Protozool. 39, 287-289. 50 ROpstorf, P. and Hiilsman, N. (19921 Sexual stages stages in the life cycle of Vampyrellidae (Rhizopoda). Eurnp. J. Prostol. 28, 355. 51 Taylor, F.J.R. (1990) Phylum Dinoflagellata. In: Margulis, L., Corliss, J.O., Melkonian, M. and Chapman, D.J., eds., Handbook of Protoctista, Jones and Bartlett Publishers, Boston, pp. 419-437. 52 Kristiansen, J. (19901 Phylum Chrysnphyta. ln: Margulis, L., Corliss, J.O., Melkonian, M. and Chapman, D.J., eds., Downloaded from https://academic.oup.com/femsre/article/15/4/321/489287 by guest on 12 July 2022 16 Chakraborty, S., Old, K.M. and Warcup, J.H. (19831 Amoebae from take-all suppressive soil which feed on Gaeumannomyces graminis tritici and other soil fungi. Soil Biol. Biochem. 15, 17-24. 17 Chakraborty, S. and Old, K.M. (1982) Mycophagous soil amoebae: Interactions with three plant pathogenic fungi. Soil Biol. Biochem. 14, 247-255. 18 Foissner, W. (1987) Soil protozoa: Fundamental problems, ecological significance, adaptations in ciliates and testaceans, bioindicators and guide to the literature. Progr. Protistol. 2, 69-212. 19 Postma, J. and Van Veen, J.A. (1990) Habitable pore space and survival of Rhizobium leguminosarum biovar trifoli introduced into soil. Microb. Ecol. 19, 149-161. 20 Febvre-Chevalier, C. (1990) Phylum Actinopoda, Class Heliozoa. In: Handbook of Protoctista (Margulis, L., Corliss, J.O,, Melkonian, M. and Chapman, D.J., Eds.), pp. 419-437. Jones and Bartlett Publishers, Boston, MD. 21 Foissner, W. (1991) Diversity and ecology of soil flagellates. In: The Biology of Free-living Heterotrophic Flagellates (Patterson, D.J. and Larsen, J., Eds.), pp. 93 112. Systematics Association, Clarendon Press, Oxford. 22 Levine, N.D., Corliss, J.O., Cox, F.E.G., Deroux, G., Grain, J., Honigberg, B,M., Leedale, G.F., Loeblich III, A.R., Lom, J., Lynn, D., Merinfeld, E.G., Page, F.C., Poljansky, G., Spraque, V., Vavra, J. and Wallace, F.G. (19801 A newly revised classification of the Protozoa. J. Protozool. 27, 37-58. 23 Lousier, J.D. and Bamforth, S.S. (1991) Soil Protozoa, In: Soil Biology Guide (Dindal, D.L., Ed.), pp. 97-136. Wiley, New York, NY. 24 Bamforth, S.S. (19801 Terrestrial protozoa. J. Protozool. 27, 404 409. 25 Laminger, H. (19781 The effects of soil moisture fluctuations on the testacean species Trinema enchelys (Ehrenberg) Leidy in a high mountain brown-earths-podsol and its feeding bchaviour. Arch. Protistenk. 120, 446-454. 26 Stout, J.D, and Heal, O.W. (1967) Protozoa In: Soil Biology (Burges, A. and Raw, F., Eds.), pp. 149-195. Academic Press, New York, NY. 27 Richards, B.N. (19871 The Microbiology of Terrestrial Ecosystems. Longman, New York, NY. 28 Van den Hoek, C. (1984) Algen. 2nd edn., Thieme, Stuttgart. 29 Kuserk, F,T. (1980) The relationship between cellular slime molds and bacteria in forest soil. Ecology 61, 14741485. 30 Feest, A. (1987) The quantitative ecology of soil mycetozoa. Prog. Protist. 2, 331-36l. 31 Page, F.C. (1976) An Illustrated Key to Freshwater and Soil Amoebae. Freshwater Biological Association, Ambleside. 32 Page, F.C. (1988) A New Key to Freshwater and Soil Gymnaboeae. Freshwater Biological Association, Ambleside. 33 Cowling, A.J. (1991) Free-living heterotrophic flagellates: methods of isolation and maintenance, including sources 348 53 54 55 51~ 58 59 611 61 (~2 63 64 65 66 67 68 69 711 71 Protozoology. Int. Congr. Ser. No. 91, p. 120. Excerpta Medicm Amsterdam. 72 Singh, B.N. (194l) Selectivity in bacterial food by soil amoebae in pure mixed culture and in sterilized soil. Ann. Appl. Biol. 28. 52 64. 73 Singh, B.N. 11945) The selection of hacterial food by soil amoebae, and tile toxic effects of bacterial pigments and other products on soil protozoa. Br. J. Exp. Pathol. 26, 316 325. 74 Anscombe, F.J. and Smgh, B,N. (1948) Limitation of bacteria by micro-predators in soih Nature 161, 140-141. 75 Singh. B.N. (1942) Toxic effects of certain bacterial metabolic products on soil protozoa. Nature 149, 16N. 7t~ Singh, B.N. (1948) Studies on giant amoeboid organisms. I. The distribution of Leptomyxa reticulata Goodcy in soils of Great Britain and tile effect of bacterial food on growth and cyst formation. J. Gen. Microbiol. 2, 8- 14. 77 Groscop, J.A. and Brent. M.M. 11964) The effects of selected stratus of pigmented microorganisms on small free-living amoebae. Can. J. Microbiol. I0, 579 584. 7,'4, Oehler, R. ( 19161 Am6benzucht auf reinem Boden. Arch. Protistenk, 37, 175-191/. 79 Upadhyay. J.M. 11968) Growth and baeteriolytic activity of a soil amoeba, ttartmanelkl glebae. J. Bacteriol, 95, 771-774. 80 Kunicki-Goldfinger, W., Drozanski, W., Blaszczak. D.. Mazur. J. and Skibinska. J. 119571 Bacterial food of soil amoebae. Acta Microbiol. Pol. 6. 331-344 (in Polish with English summary). 81 Sevcrtzova, L.B, (1928) The food requirement of soil amoebae with reference to their interrelation with soil bacteria and soil fungi. Zbl. Bakt. Parasitenkd 11. 73, 162 -179. 82 Gurijala. K.R. and Alexander, M. 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