Tropical Plant Pathology https://doi.org/10.1007/s40858-020-00344-x SHORT COMMUNICATION Is Lasiodiplodia theobromae the only species that causes leaf blight disease in Brazilian coconut palms? Pedro H. D. Santos 1 & Beatriz M. Carvalho 2 & Fernanda A. S. Aredes 2 & Vicente Mussi-Dias 1 & Danilo B. Pinho 3 & Messias G. Pereira 2 & Silvaldo Felipe da Silveira 1 Received: 5 October 2018 / Accepted: 27 March 2020 # Sociedade Brasileira de Fitopatologia 2020 Abstract Leaf blight disease in coconut (LBC) caused by Lasiodiplodia theobromae leads to early defoliation and loss of bunches, which negatively impacts coconut fruit yield. The etiology of LBC in Brazil needs to be updated based on modern tools such as DNA sequencing and phylogenetic analysis. A phylogenetic tree using ITS and TEF-1α genes in combination with morphology showed L. theobromae as the most common species associated with LBC in Brazil. We identified six species from distinct plant parts (leaves, fruits, rachis, inflorescence, stem and stipe): L. brasiliense, L. pseudotheobromae, L. laeliocattleyae, Botryosphaeria fabicerciana and B. dothidea. Our pathogenicity test results revealed that B. fabicerciana and L. pseudotheobromae cause LBC symptoms. Our study is the first to address two species, in addition to L. theobromae, as etiologic agents of LBC disease based on morphological, phylogenetic and pathogenicity data. Keywords Botryosphaeriaceae . Cocos nucifera . Bayesian inference . Taxonomy Cocos nucifera L. is the most cultivated perennial palm species in the tropics due to both its economic and social values. In Brazil, coconut producers include smallholders, family farmers and agribusinesses, and producing this crop is an important employment- and income-generating activity (Marina et al. 2009; Monteiro et al. 2013; Pereira et al. 2017). Coconut palm cultivation can generate various products and byproducts, from fruits sold directly to consumers or the supply raw materials (pulp, oil, fibers, etc.) for different uses from handicrafts to industrial uses (Mirisola Filho 2002). The coconut palm was established in Brazil in the colonial period, and in the last two decades, coconut plantation areas have been expanding to the southeastern region to supply the local * Pedro H. D. Santos pedroh_dias@hotmail.com 1 Laboratório de Entomologia e Fitopatologia, Universidade Estadual do Norte Fluminense, Campos dos Goytacazes, RJ, Brazil 2 Laboratório de Genética e Melhoramento de Plantas, Universidade Estadual do Norte Fluminense, Campos dos Goytacazes, RJ, Brazil 3 Departamento de Fitopatologia, Universidade de Brasília, Brasília, Brazil fresh fruit market with in natura coconut water for consumption. The cv. Green Dwarf of Jiqui is the most planted coconut palm variety for this purpose in Brazil due to its precocity, productivity and water quality (Mirisola Filho 2002). However, this variety is susceptible to the most damaging coconut diseases and pests. Leaf blight disease in coconut (LBC) caused by Lasiodiplodia theobromae (Pat.) Griffon & Maubl is one of the most damaging fungal diseases affecting coconut crops in Brazil (Monteiro et al. 2013). In the lower leaves of the palm, infection often occurs from the insertion points of the apical leaflets, and then, the fungus systemically (internally) invades the rachis to the petiole, causing necrosis accompanied by gum liberation and culminating in dryness of the leaflets and blight of the whole leaf (de Souza Filho et al. 1979). Consequently, a significant portion of the canopy and most of the lower leaves of the plant fall off, and the corresponding fruit bunches are lost as a consequence of physical and physiological damage. Adult Green Dwarf of Jiqui coconut palms are unproductive when they present fewer than 18 leaves (Mirisola Filho 2002). On the Brazilian southeastern coast, several coconut crops of this variety are considered unproductive because the plants have few leaves as a result of LBC outbreaks (Monteiro et al. 2013). Trop. plant pathol. LBC disease was first reported in the northeastern coastal region of Brazil by de Souza Filho et al. (1979), and then, the anamorphic specific identification of the pathogen was complemented in a more detailed taxonomic study based on morphology (Subileau et al. 1994). As occurrence of cryptic species of L. theobromae has been previously suggested (Alves et al. 2008), a review the etiology of LBC, which to date has been considered to be caused by a single species based only on morphological data (Maublanc and Griffon 1909; de Souza Filho et al. 1979; Subileau et al. 1994), needs to be conducted taking advantage of novel molecular tools and phylogenetic analysis, with higher resolution for specific classification. Using molecular data, Marques et al. (2013) identified eight Lasiodiplodia species associated with peduncular rot in mango, which was previously attributed only to L. theobromae based on morphology. In this study, we tested the hypothesis that LBC in Brazil is not caused by a single species but by a complex of species. To conduct this study, morphological, phylogenetic and pathogenicity data were collected and analyzed for the first time for this pathosystem, to the best of our knowledge. Our hypothesis supports previous work on postharvest stem-end rot of coconut in Brazil associated with four Lasiodiplodia species based on morphology and phylogenetic analyses of DNA sequences (Rosado et al. 2016). For fungal isolation, 90 LBC-symptomatic samples were collected from coconut palms and classified according to geographic origin (Brazilian States) and plant organ (leaves = 9, fruits = 9, rachis = 67, inflorescence = 1, stem = 1 and stipe = 1). Two samples of other hosts displaying disease symptoms were collected. Pathogen isolation was carried out based on the method by Ismail et al. (2012). All cultures were stored in Petri dishes at −20 °C. The Lasiodiplodia isolates were cultured in potato dextrose agar (PDA) medium (Sigma-Aldrich). From the samples, 23 isolates matched Lasiodiplodia sp. characteristics, based on morphology and cultural appearance. Then, 22 isolates were obtained from symptomatic organs, and only one isolate was originally endophytic (CF/ UENF429), i.e., it was isolated without symptoms. With respect to the plant organs of origin, four isolates were obtained from leaves, seven from fruits, and nine from rachises, and the remaining three isolates were from inflorescences, stems and stipes (one from each organs). Most isolates were collected in Rio de Janeiro state (10 isolates), followed by collections in Pernambuco (8), Pará (2), Bahia (1), Alagoas (1) and Sergipe (1) states. For DNA extraction, approximately 0.1 g of mycelia from pure cultures was macerated inside tubes containing electromagnetic beads and 600 μL of nuclei lysis solution. The tubes were placed three times in a cell disruptor (Loccus L-Beader 3) at 3700 rpm/40 s. DNA extraction steps implemented after the maceration procedure followed the protocol by Pinho et al. (2013), and we used the Promega DNA purification kit (WizardGenomic DNA Purification Kit). The internal transcribed spacer (ITS) was amplified with primers ITS1 and ITS4 (White et al. 1990) and translation elongation factor 1alpha (TEF 1-α) with primers EF1-728F and EF2 (Jacobs et al. 2004). PCR conditions were the same as those reported by Santos et al. (2017). Samples were sent for sequencing to ACTGene Análises Moleculares Ltda. (Biotechnology Center, Federal University of Rio Grande do Sul, Porto Alegre, RS, Brazil). We edited the nucleotide sequences in DNA Dragon software (Hepperle 2011). All sequences were manually corrected, and nucleotide arrangements, at ambiguous positions, were corrected by primer sequences in directions 5′-3 ‘and 3’-5′. The new sequences were deposited in GenBank (Table 1). Additional sequences of ITS and TEF-1α regions were downloaded from GenBank. Consensus regions were compared in the GenBank database to check molecular identification through the nucleotide BLAST algorithm (Altschul et al. 1990). Two isolates were classified as Botryosphaeria; the other isolates were Lasiodiplodia; therefore, each genus was subjected to separate phylogenetic analyses. MUSCLE was used in the alignments (Edgar 2004), and then, the alignments were manually checked and improved in MEGA v.7, whenever necessary (Kumar et al. 2016). The assessed gene regions were concatenated in the software Mesquite v.3.40 (Maddison and Maddison 2018). Bayesian inference (BI) analysis was carried out based on the Monte Carlo Markov chain method (MCMC). The MrMODELTEST v. 3.04 (Posada and Buckley 2004) was adopted to select the nucleotide substitution model for BI analysis. Models were estimated for each gene region separately. Likelihood values were calculated, and the model was selected according to the Akaike information criterion (AIC). Evolution models selected for each gene region were HKY + I (Botryosphaeria spp.) and K80+ I (Lasiodiplodia spp.) for ITS and HKY + G (Botryosphaeria spp.) and GTR + I + G (Lasiodiplodia spp.) for TEF1-α. The BI analysis was carried out in MrBayes v.3.1.1 (Ronquist and Huelsenbeck 2003). The consensus tree was obtained after 10 million generations of a Markov chain were applied to two runs, four chains each, with a 25% burnin. Likelihood log convergence was analyzed in TRACER v. 1.4.1 software (Rambaut and Drummond 2013). The tree was visualized in FigTree v. 1.4.4 (Rambaut 2009) and exported to graphic software. We chose L. crassispora as the outgroup for the analysis of Lasiodiplodia sp. since this species is basal to all Lasiodiplodia species. For Botryosphaeria analysis, Neofusicoccum pennatisporum was used as the outgroup. The Lasiodiplodia isolates from coconut were grouped into four main clades (Fig. 1), whereas the Botryosphaeria isolates were grouped into two clades (Fig. 2). Most isolates (16) formed the largest clade, which corresponded to the species L. theobromae, which recorded a high posterior probability value (pp = 0.96) (Fig. 1). This outcome confirms the worldwide distribution of this pathogen, as Marques et al. (2013) Trop. plant pathol. Table 1 Collection details and GenBank accession numbers of isolates included in the present study Species Lasiodiplodia theobromae Culture accession number CBS 164.96 CBS 124.13 CF/UENF428c CF/UENF427 CF/UENF421 CF/UENF423 CF/UENF429 Host Location Genbank ITSa TEF 1-αb Unidentified fruit along coral reef coast – Cocos nucifera Persea americana Cocos nucifera Cocos nucifera Capparis flexuosa Papua New Guinea USA Brazil Brazil Brazil Brazil Brazil AY640255 AY640258 EU673195 KY655194 KY655193 KY655200 KY655202 KY655195 DQ458875 KY223711 KY223707 KY223708 KY223710 KY223712 CF/UENF438 CF/UENF430 CF/UENF432 CF/UENF425 CF/UENF422 CF/UENF435 CF/UENF431 CF/UENF420 CF/UENF426 CF/UENF437 CF/UENF419 CBS110492 CMW22653 CBS122065 CBS122519 CMM2255 CMM4015 COAD 1784 Cocos nucifera Cocos nucifera Cocos nucifera Elaeis guineensis Cocos nucifera Cocos nucifera Cocos nucifera Cocos nucifera Cocos nucifera Cocos nucifera Cocos nucifera – Pterocarpus angolensis Adansonia gibbosa Adansonia gibbosa Carica papaya Mangifera indica Brazil Brazil Brazil Brazil Brazil Brazil Brazil Brazil Brazil Brazil Brazil – South Africa Australia Australia Brazil Brazil KY655211 KY655203 KY655205 KY655191 KY655201 KY655208 KY655204 KY655199 KY655192 KY655210 KY655198 EF622086 FJ888465 EU144051 EU144050 KC484792 JX464063 KY223717 KY223713 KY223715 KY223705 KY223709 KY223716 KY223714 KY223720 KY223706 KY223718 KY223719 EF622066 FJ888452 EU144066.1 EU144065 KC481523 JX464049 Cocos nucifera Brazil KP244693 KP308469 COAD 1786 CF/UENF436 CF/UENF439 UCD2553AR UCD2604MO BL104 BL185 CMW27801 CMW27820 BOT10 BOT29 CF/UENF417 IRAN1521C IRAN1522C CMM3609 CMM3652 IRAN1498C IRAN1500C Cocos nucifera Cocos nucifera Cocos nucifera – – Genista monspessulana Genista monspessulana Terminalia catappa Terminalia catappa Mangifera indica Mangifera indica Cocos nucifera – – Jatropha curcas Jatropha curcas – – Brazil Brazil Brazil – – Tunisia Tunisia Madagascar Madagascar Egypt Egypt Brazil – – Brazil Brazil – – KP244696 KY655209 KY655212 HQ288227 HQ288228 KJ638317 KJ638319 FJ900595 FJ900597 JN814397 JN814401 KY655196 GU945353 GU945354 KF234543 KF234554 GU945356 GU945355 KP308471 KY223705 KY223719 HQ288269 HQ288270 KJ638336 KJ638338 FJ900641 FJ900643 JN814424 JN814428 KY223714 GU945339 GU945340 KF226689 KF226715 GU945344 GU945343 Lasiodiplodia parva CBS 495.78 CBS 456.78 Manihot esculenta Manihot esculenta Colombia Colombia KX464632 EF622063 Lasiodiplodia mediterranea BL1 Quercus ilex Italy KX464138 NR_ 111265 KJ638312 Lasiodiplodia crassispora Lasiodiplodia margaritacea Lasiodiplodia brasiliensis Lasiodiplodia viticola Lasiodiplodia exigua Lasiodiplodia mahajangana Lasiodiplodia laeliocattleyae Lasiodiplodia citricola Lasiodiplodia euphorbicola Lasiodiplodia hormozganensis KJ638331 Trop. plant pathol. Table 1 (continued) Species Lasiodiplodia subglobosa Lasiodiplodia pseudotheobromae Lasiodiplodia iraniensis Lasiodiplodia gilanensis Lasiodiplodia venezuelensis Botryosphaeria fabicerciana Botryosphaeria dothidea Botryosphaeria fusispora Botryosphaeria corticis Botryosphaeria scharifii Botryosphaeria ramosa Botryosphaeria agaves Culture accession number Host Location Genbank ITSa TEF 1-αb BL101 CMM3872 CMM4046 CBS116459 CMM3887 CF/UENF434 CF/UENF433 IRAN1517C IRAN1519C IRAN1501C IRAN1523C WAC12539 CMW13513 CMW27094 CMW 27108 CF/UENF418 CMM3937 CMM3938 Vitis vinifera Jatropha curcas Jatropha curcas Gmelina arborea Jatropha curcas Cocos nucifera Cocos nucifera – – – – Acacia mangium Acacia mangium Eucalyptus sp. Eucalyptus sp. Cocos nucifera Mangifera indica Mangifera indica Italy Brazil Brazil Costa Rica Brazil Brazil Brazil – – – – Venezuela Venezuela China China Brazil Brazil Brazil KJ638311 KF234558 KF234560 EF622077 KF234559 KY655207 KY655206 GU945349 GU945350 GU945352 GU945351 DQ103547 DQ103549 HQ332197 HQ332200 KY655197 JX513643 JX513645 KJ638330 KF226721 KF226723 EF622057 KF226722 KY223718 KY223721 GU945337 GU945338 GU945341 GU945342 DQ103568 DQ103570 HQ332213 HQ332216 KY223714 JX513622 JX513624 CF/UENF416 MFLUCC 10–0098 MFLUCC 11–0507 CBS 119047 ATCC 22927 IRAN1529C Cocos nucifera Entada sp. Caryota sp. Vaccinium corymbosum Vaccinium sp. Brazil Thailand Thailand USA USA KY655190 JX646789 JX646788 DQ299245 DQ299247 KY223723 JX646854 JX646853 EU017539 EU673291 Mangifera indica Mangifera indica Eucalyptus camaldulensis Agave sp. Agave sp. Iran Iran Australia Thailand Thailand JQ772020 JQ772019 EU144055 JX646791 JX646790 JQ772057 JQ772056 EU144070 JX646856 JX646855 IRAN1543C CBS 122069 MFLUCC 11–0125 MFLUCC 10–0051 a ITS = internal transcribed spacer; b TEF1-α = translation elongation factor 1-α. c Isolates and newly generated sequences are highlighted in bold. CF/ UENF = Collection of the Plant Disease Clinic of Darcy Ribeiro Northern Rio de Janeiro State University - UENF and Rosado et al. (2016) have reported. We observed the formation of two clades of L. brasiliense and L. pseudotheobromae, with two isolates in each clade (pp = 0.97 and 0.99, respectively). One isolate was observed in the L. laeliocattleyae clade (pp = 0.99) (Fig. 1). There were some isolates in the B. fabicerciana and B. dothidea clades (both with pp. = 0.96) (Fig. 2). Lasiodiplodia theobromae isolates originated from different plant organs and Brazilian states. Isolate CF/UENF416 (B. dothidea) was collected in the stipe samples of the asymptomatic plants that were harvested in northern Rio de Janeiro state. The CF/UENF417 and CF/UENF418, both classified as L. laeliocattleyae, and the B. fabicerciana isolates were collected in the fruits sampled in Bahia and in northern Rio de Janeiro state, respectively. CF/UENF433, CF/UENF434 (L. pseudotheobromae) and CF/UENF436 (L. brasiliense) were isolated from coconut rachises in Pernambuco state and presented blight symptoms. CF/UENF439 (L. brasiliense) was isolated from the necrotic inflorescences of plants harvested in Alagoas state. Mycelial growth, colony staining, and conidial morphology were measured after incubation at 28 ± 1 °C under a 12 h photoperiod. Mycelial growth was evaluated through colony diameter measurements taken in two orthogonal directions, every 12 h, to determine its mean from four replicates (9 cm diameter Petri dish). Evaluations were completed when the first isolate reached the Petri dish border since this factor determined the mean fungal growth rate (mm/day). The prevailing colony color was observed 15 days after incubation, based on the descriptive scale reported by Lima et al. (2013). This experiment followed a completely randomized design (CRD) with four replicates, with each plate being the experimental Trop. plant pathol. Fig. 1 Phylogram based on the Bayesian Inference of two combined sequences (ITS and TEF 1-α) of Lasiodiplodia isolates associated with Cocos nucifera. The posterior probability is indicated near the branch nodes. The tree was rooted in L. crassipora unit. Data were subjected to analysis of variance (ANOVA), and the means were grouped through the Scott-Knott test (p = 0.05), which was conducted in R software (R Development Core Team 2015) with the ExpDes package (Ferreira et al. 2014). There were significant differences between the isolates and species in terms of the mean mycelial growth rates and colony staining variation in the cultivated fungus population (Table 2). CF/UENF421 was the first isolate to reach the Petri Trop. plant pathol. Fig. 2 Phylogram based on the Bayesian Inference of two combined sequences (ITS and TEF 1-α) of Botryosphaeria isolates associated with Cocos nucifera. The posterior probability is indicated near the branch nodes. The tree was rooted in Neofusicoccum pennatisporum dish border within 48 h. Isolates showed mycelial growth rates of 87 mm/day ±0.19 (L. brasiliense), 78.2 mm/day ±0.38 (L. laeliocattleyae), 72 mm/day ±0.18 (L. theobromae), 73.9 mm/day ±0.34 (L. pseudotheobromae), 44.7 mm/day ±0.12 (B. dothidea) and 35.2 mm/day ±0.29 (B. fabicerciana) (Table 2). Colony staining ranged from light gray to dark gray and black. The clusters of isolates based on mycelial growth and colony staining differed among the isolates, regardless of the species. Most isolates in our study presented light gray colonies (Table 2). To determine the pathogenicity of the isolates, we inoculated coconut palm rachises (four rachises in six plants) without visible symptoms of the disease, with PDA discs containing the mycelium of each representative species. Mycelium was used instead of conidia because some isolates had little sporulation or produced sterile pycnidia. Symptom emergence was monitored through 30 days of incubation. The first symptoms appeared on approximately the 11th day. All LBC symptoms caused by the B. fabicerciana and L. pseudotheobromae isolates were identified during the pathogenicity test carried out with the coconut palms (Fig. 2). We observed distinct types of symptoms induced by B. fabicerciana and L. pseudotheobromae. In comparison to L. pseudotheobromae, B. fabicerciana isolates were more efficient, causing more visible internal symptoms. This is the first study to address B. fabicerciana and L. pseudotheobromae as causal agents of leaf blight disease in coconut Fig. 3. The six species clustered in a well-supported clade with other isolates analyzed in previously published studies (Alves et al. 2008; Ismail et al. 2012; Phillips et al. 2013; Netto et al. 2014; Rosado et al. 2016). The higher prevalence of L. theobromae among isolates was expected due to its known cosmopolitan distribution and wide range of hosts (Punithalingam 1980; Burgess et al. 2006; Marques et al. 201; Netto et al. 2014). Our results, as well as those recorded by Marques et al. (2013) and Netto et al. (2014), indicate that L. laeliocattleyae and L. pseudotheobromae are also frequent species associated with coconut disease. L. theobromae, L. pseudotheobromae, and B. fabicerciana were also pathogenic in coconut rachises and were also associated with postharvest stem-end rot in coconut fruits (Rosado et al. 2016s). Trop. plant pathol. Table 2 Mycelial growth rate and colony staining of pathogenic coconut isolates (Cocos nucifera) Isolate Species Growth (cm/day)* Colony staining *** CF/UENF428 CF/UENF436 CF/UENF427 CF/UENF421 CF/UENF423 L. theobromae L. brasiliense L. theobromae L. theobromae L. theobromae 8.95 A** 8.75 A 8.67 A 8.61 A 8.61 A DG DG LG LG LG CF/UENF429 CF/UENF433 CF/UENF438 CF/UENF430 CF/UENF432 CF/UENF417 CF/UENF425 CF/UENF422 CF/UENF439 CF/UENF435 CF/UENF431 CF/UENF434 CF/UENF420 CF/UENF426 CF/UENF416 CF/UENF437 CF/UENF418 CF/UENF419 L. theobromae L. pseudotheobromae L. theobromae L. theobromae L. theobromae L. laeliocattleyae L. theobromae L. theobromae L. brasiliense L. theobromae L. theobromae L. pseudotheobromae L. theobromae L. theobromae B. dothidea L. theobromae B. fabicerciana L. theobromae 8.50 A 8.46 A 8.38 A 7.88 B 7.88 B 7.82 B 7.70 B 7.62 B 7.40 B 7.17 C 7.08 C 6.33 D 5.88 E 5.75 E 4.47 F 3.95 G 3.52 G 2.70 H BL LG LG LG LG BL LG LG DG DG DG LG LG DG BL DG DG DG * Means of 4 replicates per treatment. Means followed by the same letter in the column did not differ from each other in the Scott-Knott test at 5% significance level; *** LG Light gray, DG Dark gray, BL Black Variations in mycelial growth rates and colony staining data were within the limits described in other studies for all corresponding species of Lasiodiplodia identified in our study Fig. 3 Disease symptoms on coconut tree rachis inoculated with pathogenic fungal isolates. a - Lasiodiplodia pseudotheobromae; b Botryosphaeria fabicerciana and c - Internal symptoms (Alves et al. 2008; Abdollahzadeh et al. 2010; de Lins et al. 2010; Freire et al. 2013; Machado et al. 2014), and these characteristics do not seem to discriminate the Lasiodiplodia Trop. plant pathol. spp. Here, we reinforce the need for molecular and phylogenetic studies to resolve identification problems, especially in groups where cryptic species such as Lasiodiplodia occur. Morphology data were also not sufficient to discriminate the species explaining the single-species hypothesis. In fact, identification of Botryosphaeriaceae using morphology only allows the separation of isolates to the genus level, with DNA phylogeny being the most relevant tool for describing Lasiodiplodia species according to the phylogenetic species concept (Ismail et al. 2012; Phillips et al. 2013; Slippers et al. 2014; Osorio et al. 2017). Within Botryosphaeriaceae, the use of DNA sequence data from multiple loci is strongly recommended (Phillips et al. 2013; Slippers et al. 2014; RodríguezGálvez et al. 2017; Rosado et al. 2016; Bautista-Cruz et al. 2019). Our study is the first to address B. fabicerciana and L. pseudotheobromae in addition to L. theobromae as causal agents of leaf blight disease in coconut based on phylogenetic, morphological and pathogenic data. 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