Morpho-Molecular Identification and Pathogenic Characterization of Fusarium and Colletotrichum Species Associated with Intercropped Soybean Pod Decay

The fruiting stage of soybean (<i>Glycine max</i> L.) is critical for determining both its yield and quality, thereby influencing global production. While some studies have provided partial explanations for the occurrence of <i>Fusarium</i> species on soybean seeds and pods, the fungal diversity affecting soybean pods in Sichuan Province, a major soybean cultivation region in Southwestern China, remains inadequately understood. In this study, 182 infected pods were collected from a maize-soybean relay strip intercropping system. A total of 10 distinct pod-infecting fungal genera (132 isolates) were identified, and their pathogenic potential on soybean seeds and pods was evaluated. Using morphological characteristics and DNA barcode markers, we identified 43 <i>Fusarium</i> isolates belonging to 8 species, including <i>F. verticillioides</i>, <i>F. incarnatum</i>, <i>F. equiseti</i>, <i>F. proliferatum</i>, <i>F. fujikuroi</i>, <i>F. oxysporum</i>, <i>F. chlamydosporum</i>, and <i>F. acutatum</i> through the analysis of the translation elongation factor gene (<i>EF1-α</i>) and RNA polymerases II second largest subunit (<i>RPB2</i>) gene. Multi-locus phylogenetic analysis, incorporating the Internal Transcribed Spacer (<i>rDNA ITS</i>), β-tubulin (<i>β-tubulin</i>), Glyceraldehyde 3-phosphate dehydrogenase (<i>GADPH</i>), Chitin Synthase 1 (<i>CHS-1</i>), Actin (<i>ACT</i>), Beta-tubulin II (<i>TUB2</i>), and Calmodulin (<i>CAL</i>) genes distinguished 37 isolates as 6 <i>Colletotrichum</i> species, including <i>C. truncatum</i>, <i>C. karstii</i>, <i>C. cliviicola</i>, <i>C. plurivorum</i>, <i>C. boninense</i>, and <i>C. fructicola</i>. Among these, <i>F. proliferatum</i> and <i>C. fructicola</i> were the most dominant species, representing 20.93% and 21.62% of the isolation frequency, respectively. Pathogenicity assays revealed significant damage from both <i>Fusarium</i> and <i>Colletotrichum</i> isolates on soybean pods and seeds, with varying isolation frequencies. Of these, <i>F. proliferatum</i>, <i>F. acutatum</i>, and <i>F. verticillioides</i> caused the most severe symptoms. Similarly, within <i>Colletotrichum</i> genus, <i>C. fructicola</i> was the most pathogenic, followed by <i>C. truncatum</i>, <i>C. karstii</i>, <i>C. cliviicola</i>, <i>C. plurivorum</i>, and <i>C. boninense</i>. Notably, <i>F. acutatum</i>, <i>C. cliviicola</i>, <i>C. boninense</i>, and <i>C. fructicola</i> were identified for the first time as pathogens of soybean pods under the maize-soybean strip intercropping system in Southwestern China. These findings highlight emerging virulent pathogens responsible for soybean pod decay and provide a valuable foundation for understanding the pathogen population during the later growth stages of soybean.