Identification and Comparison of Colletotrichum Secreted Effector Candidates Reveal Two Independent Lineages Pathogenic to Soybean

Colletotrichum is one of the most important plant pathogenic genus of fungi due to its scientific and economic impact. A wide range of hosts can be infected by Colletotrichum spp., which causes losses in crops of major importance worldwide, such as soybean. Soybean anthracnose is mainly caused by C. truncatum, but other species have been identified at an increasing rate during the last decade, becoming one of the most important limiting factors to soybean production in several regions. To gain a better understanding of the evolutionary origin of soybean anthracnose, we compared the repertoire of effector candidates of four Colletotrichum species pathogenic to soybean and eight species not pathogenic. Our results show that the four species infecting soybean belong to two lineages and do not share any effector candidates. These results strongly suggest that two Colletotrichum lineages have acquired the capability to infect soybean independently. This study also provides, for each lineage, a set of candidate effectors encoding genes that may have important roles in pathogenicity towards soybean offering a new resource useful for further research on soybean anthracnose management.

Identification and Comparison of Colletotrichum Secreted Effector Candidates Reveal Two Independent Lineages Pathogenic to Soybean

Colletotrichum is one of the most important plant pathogenic genera of fungi due to its scientific and economic impact. Colletotrichum spp. can infect a wide range of hosts, causing losses in crops of major importance worldwide, such as soybean. In the past, soybean anthracnose was mainly caused by C. truncatum, but during the last decade, other species have been identified at an increasing rate, becoming one of the most important limiting factors to soybean production in several regions. To gain a better understanding of the evolutionary origin of soybean anthracnose, we compared the repertoire of effector candidates of four Colletotrichum species pathogenic to soybean and eight pathogens of other hosts. Our results show that the four species infecting soybean belong to two lineages and do not share any of the lineage specific effector candidates identified. These results strongly suggest that two Colletotrichum lineages have acquired the capability to infect soybean independently. This study also provides, for each lineage, a set of candidate effectors encoding genes that may have important roles in pathogenicity towards soybean offering a new resource useful for further research on soybean anthracnose management.

First Report of Colletotrichum brevisporum Causing Soybean Anthracnose in China

In March 2020, widespread anthracnose was observed on soybean (Glycine max) in southeastern Jiangsu (Nantong municipality; 120.53° E, 31.58° N) in China. Plants exhibited irregular brown necrotic lesions in stem and leaves, and pronounced wilting. The symptoms were detected in one soybean field, 0.42 ha, surrounded by healthy wheat fields. Approximately 65% of the soybean plants showed the disease symptoms, and crop yield was reduced by 28-35% with respect the yield achieved in previous years, when no symptoms were observed. The symptoms were consistent with those previously reported for anthracnose on soybean caused by Colletotrichum chlorophyti, C. cliviae and C. gloeosporioides (Barbieri et al. 2017; Mahmodi et al. 2013; Yang et al. 2012). Diseased, 3-week old plants were collected. Small pieces, approximately 1 cm2 in size, of symptomatic tissue were surface sterilized in 1.5% NaOCl for 1 min, and washed twice with sterile ddH2O. The pathogen was isolated and cultured on potato dextrose agar (Song et al. 2020), containing chloramphenicol (50 µg/mL), under darkness at 28 °C for 3 days. Sequence of internal transcribed spacer (ITS), actin (ACT), β-tubulin (TUB2) and glyceraldehyde 3-phosphate dehydrogenase (GAP/span>DH) genes was performed as reported by Yang et al. (2015). Sequences were submitted to GenBank under accession numbers MT361074 (ITS) and MT415548-MT415550 (ACT, TUB2 and GAPDH). Blast search revealed that the amplified sequences had 100% (ITS; C. brevisporum TCHD, MH883805), 97.66% (ACT; C. brevisporum S38, KY986905), 99.06% (TUB2; C. brevisporum PF-2, KY705061) and 100% (GAPDH; C. brevisporum LJTJ27, KP823797) matches to multiple C. brevisporum strains, whereas all reported C. chlorophyti, C. cliviae and C. gloeosporioides strains showed no similarity to at least 2 of the studied genes. Molecular phylogenetic tree constructed using MEGA7 confirmed the identity of the pathogen. ACT and ITS sequences were blasted separately in Muscle (https://www.ebi.ac.uk/Tools/msa/muscle/) and then combined together to make the phylogenetic tree. The evolutionary history was inferred by using the Maximum Likelihood method based on the Tamura 3-parameter model, and the tree with the highest log likelihood (-1749.2186) is shown in Figure 1. The Colletotrichum strains previously found causing anthracnoseon soybean, and other relevant strains used in taxonomic analyses were included in the phylogenetic tree. Microscope observations showed the presence of 15-µm-long cylindrical conidia and septate mycelium, and agree with those reported for the morphology of C. brevisporum by Damm et al. (2019). To confirm pathogenicity, the mycelia from a 2 day-old culture on PDA was collected and suspended in sterile ddH2O (≈ 106 cells/mL) to prepare the inoculum. The pathogen was sprayed-inoculated on stem and leaves of healthy soybean plants. In control plants, sterile ddH2O was used. Inoculated plants were maintained in growth chamber at 28 °C and 50% relative humidity. Typical anthracnose symptoms were obsered 20 days after inoculation (Figure 2). C. brevisporum was reported to produce anthracnose on pumpkin, papaya, mulberry, coffee, passion fruit and pepper in China (Liu et al. 2017; Liu et al. 2019; Xue et al. 2019). Here, we report for the first time C. brevisporum causing anthracnose on soybean, an economically-relevant crop in China.

Current Status of Soybean Anthracnose Associated with Colletotrichum truncatum in Brazil and Argentina

Brazil and Argentina have a combined soybean area of 53.6 million hectares, which accounts for over half of the total global production. The soybean crop in South America extends from latitude 8–10° S to 32–36° S. Such a vast, almost contiguous area imposes a serious sanitary risk to the crop. Currently, the prevalence of anthracnose is increasing, with recurring reports of severe epidemics and expressive yield losses. Soybean anthracnose is mainly associated with Colletotrichum truncatum, although other Colletotrichum species have also been reported as causal agents of this disease. Knowledge about the morphological, cultural, and molecular variability of C. truncatum in South America is crucial for disease management. Here, we present data on the molecular, morphological, biological, cultural, and pathogenicity of C. truncatum isolates collected in Brazil and Argentina. Light microscopy and randomly-amplified polymorphic DNA (RAPD) analysis were used for estimating the variability of isolates. Colletotrichum truncatum displayed three types of conidiogenesis, viz. conidial formation from conidiogenous cells on hyphal extremities, in conidiomas in acervuli, and directly from fertile setae (a mechanism yet-unreported for C. truncatum). RAPD profiling was effective in revealing the genetic diversity among C. truncatum isolates. The intra-group similarity was greater among the Argentinian isolates when compared to the Brazilian group. Furthermore, the results indicated a strong correlation between geographical origin and molecular grouping, with the exclusive or semi-exclusive assembling of Brazilian and Argentinian isolates in distinct clades. Finally, a preliminary account of the reaction of soybean accessions to C. truncatum is also included.