Diversity of the bean anthracnose pathogen, colletotrichum lindemuthianum (sacc. and magn.) from major bean agroecological zones of Uganda

Abstract

Anthracnose disease in common bean (Phaseolus vulgaris L.) is a major disease caused by the fungus Colletotrichum lindemuthianum. Management through host-plant resistance is the best management option, but is constrained by the high variability among C. lindemuthianum isolates. Therefore, understanding pathogen diversity is a prerequisite to developing varieties with durable resistance. This study therefore, sought to understand the cultural, morphological, phenotypic and genetic diversity of C. lindemuthianum isolates from six major bean agroecological zones of Uganda. Diseased samples were randomly collected from two sub-counties per district from seven farmers’ fields distanced by 5-10km. To isolate the pathogen, diseased bean pods and or leaves showing symptoms of anthracnose disease were cleaned with sterile water then blot dried on sterile tissue papers. The infected tissues were cultured on potato dextrose Agar (PDA) media plates. Emerging fungi (whitish grey) suspected to be C. lindemuthianum were sub-cultured and used for diversity studies. Mycelial plugs (5mm) of each of 130 purified C. lindemuthianum isolates was placed in middle of freshly prepared Potato Dextrose Agar (PDA) media plates, in a completely randomized design with three replicates per isolate. Cultures were incubated in the dark at 25º C until the appearance of the first fungal growth. Colony growth rates were measured in an interval of 2 days until the entire plate was covered. The colony colour and edge were examined against a white background following the mycological color chart. Size and shapes of five conidia, were determined from 14 daysold cultures using the X40 magnification with the aid of Ocular (Amscope) and stage micrometer on a compound microscope. A total of 112 C. lindemuthianum isolates, grouped into five morphological groupings were used to identify races of the pathogen using the 12 International Center for Tropical Agriculture (CIAT) differential cultivars and two susceptible checks. Four plants replicated in a plastic pot were inoculated with each isolate and disease reaction evaluated after 7 days using the CIAT scale of 1-9. Races of the pathogen were classified using the binary nomenclature system. To determine genetic diversity, DNA was extracted using a modified Cetyltrimethyl Ammonium Bromide (CTAB) protocol. Two Mitochondrial Cytochrome oxidase subunit 1 (mtCOX1) gene sequences was used to analyse the genetic differentiation and haplotype diversity of C. lindemuthianum. Two mtCOX1 forward primers, SEQ1 and SEQ2 were designed using Primer3 from mitochondrial sequence (GenBank under accession: KF953885). The PCR products were sequenced at Macrogen. DnaSP (Version 6) software was used to generate population genetics parameters, and MEGA7 to align and infer maximum likelihood phylogenetic tree. For cultural and morphological variability, colony surface color ranged from light grey, grey, dark grey to black colony with varying edges; Including lobate, undulate, circular and irregular. Macro-conidia were hyaline, single celled, elongated / cylindrical or sickle shaped, with some isolates having conidia that were slightly pointed at one end. The size of conidia ranged between 12.5-15 µm long and 4.75-5.0 µm wide. Growth rate of C. lindemuthianum isolates was not significantly different on day 3, but varied significantly on days 5, 7, 9 to 11. The study identified 51 races of C. lindemuthianum from 28 districts of Uganda. Lake Victoria crescent region had the highest number of races (28 races), followed by Southwestern highland zone (23 races). Andean race 2 and middle Mesoamerican race 66 were the most prevalent races and widely distributed throughout the agroecological zones. Thirty-eight new races were observed in Uganda for the first time. Southwestern highland zone had the highest number of new races (10), followed by Lake Victoria crescent (8), Eastern highland zone (4) and 14 races were found distributed in more than one agroecological zone. Fifty-eight (58) sequences, 1147 nucleotides in length were concatenate by Geneious software. The haplotype diversity of each C. lindemuthianum populations ranged from 0.45 (Southwestern highland zone) to 0.8 (Eastern highland zone). The nucleotide diversity within C. lindemuthianum population was low and ranged from 0.052% (Northern mixed farming system zone) to 0.116% (Eastern highland zone). Nucleotide diversity between C. lindemuthianum populations was low and ranged from 0.065% to 0.091%. The study revealed a weak genetic differentiation (FST = 0.00932) between C. lindemuthianum populations. This study showed that C. lindemuthianum causing bean anthracnose in Uganda is morphologically and phenotypically diverse but genetically similar. Virulence caused by this pathogen can best be studied using the phenotypic race evaluation. Seven races (863, 985, 3663, 4033, 4039, 4041, and 4044) were considered the most virulent based on the number of differential cultivars infected. Differential cultivars with the broadest resistance indices included Kaboon (93.7%), PI207262 (89.3%), TO (89.3%), G2333 (89.3%), AB136 (87.5%) and Widusa (86.6%), and therefore recommended for use in bean breeding program for anthracnose resistance.