Characterization of southern African Colletotrichum lindemuthianum isolates using neutral and selective genetic markers

Bean anthracnose (Colletotrichum lindemuthianum) is an important disease of common beans in Zambia, and in many tropical regions of the world. Epidemics may occur in cool, wet and humid regions of the world where susceptible cultivators are grown. The disease attacks all the above ground parts of the bean plant and, if not controlled, has the potential to cause up to 90% yield loss. The causal agent of (Colletotrichum lindemuthianum) is a hyper-variable fungus with a high potential to overcome resistance genes. World over, ten resistance genes from the standard set of Centro International de Agricultural Tropical differential cultivators are being deployed in various combinations in an attempt to manage the pathogen. However, because the pathogen is highly variable it is important to monitor the incidence and severity of the disease as well as genetic variability of the pathogen in the target locality. This provides information on the potential of the pathogen to evolve as well as an indication of the important resistance genes for pyramiding. This information remains unknown in Zambia and Southern African as a whole. Thus, the goal of this research undertaking was to study the occurrence of bean anthracnose epidemics and characterize C.lindemuthianum populations, in Zambia and Southern Africa. Specifically, the study sought to assess the severity and incidence of anthracnose in bean growing regions in Zambia and assess the genetic variability of Southern Africa derived Collectotrichum lindemuthianum isolates using Simple Sequence Repeats (SSR), Random Amplified Microsatellites (RAM) and race differentials. The study was conducted using samples collected from four bean growing districts representing different agro-ecological zones in Zambia. These were Kasama, Mbala, Solwezi and Kabwe. In addition, the districts differed in farming systems, major vegetation cover and weather conditions. A hierarchical sampling structure was used at three levels i.e., agro-ecological zones, districts within agro-ecological zones and fields within districts to obtain disease bean leaf samples from the different locations. Disease incidence was assessed by expressing the proportion of diseased plants along a transect as a percentage of the total and severity was rated on a scale of 1-9, where 9 = high and 1 = low level of disease. DNA of isolates was amplified with SSRs and RAMs. The PCR products were separated by agarose gel electrophoresis. And data was scored using a binary of 0 or 1 where, 0 = no amplification and 1 = amplification. Field incidence and severity data were subjected to nested analysis of variance. Anthracnose disease maps were constructed using the geographic information systems software. The binary data were subjected to analysis of molecular variation (AMOVA). Twenty isolates were tested for their virulence against the CIAT standard set of race differential cultivators. Disease reaction was rated on a 1-9 severity scale, where 1-3 is resistant and 4-9 is susceptible reactions respectively. The sum of the binary numbers for the susceptible cultivators gave the race name. The results showed a significant agro-ecological effect (P≥ 0.001) on both incidence and severity of anthracnose in Zambia. This could be a reflection of the effect of different weather conditions and cropping patterns on bean anthracnose epiphytotic. Cool, wet and humid regions of agro-ecological zones III had higher incidence (38.1%) and severity (4.2) of the disease. Similar patterns were observed with the disease maps. SSR and RAM techniques produced 20-40 and 30-55 polymorphic loci, respectively. Phonetic analysis using the Neighbour-joining algorithm generated only one major cluster. However, there was a tendency for isolates of similar geographic origin to cluster together. AMOVA of both SSR and RAM data revealed the presence of a population structure (SSR: ᶲFST = 0.17, P = 0.001; RAM: ᶲ FST = 0.13, P = 0.001) indicating a presence of population differential in Southern Africa. The results of the AMOVA also revealed that the allelic variation (SSR = 82.97 %, RAM = 87.45%) was shared within populations. Average gene diversity over all loci ranged from 0.17-0.35 for SSRs and 0.25 to 0.36 for RAMSs showing high diversity within isolates. From the 20 isolates tested on race differential cultivars, 17 physiological races were identified, consistent with the high molecular variability revealed by molecular marker analysis. Two cultivars P1 207262 and Tu both succumbed to only one isolate, making them good candidates for resistance breeding. Highly virulent strains were also identified (2252 and 3593). Overall the high prevalence, incidence and severity of the anthracnose epidemic in agro-ecology III of Zambia and the presence of highly virulent races of C.lindemuthianum show that bean anthracnose is a major threat to bean production in the region and the greater Southern Africa. The high variability is indicative of its high potential to evolve. Host resistance breeding efforts should target pyramiding genes Co-43, Co-9 (both from PI 2071262) and Co-5 (from cultivator Tu) into locally adapted bean varieties.