Infection of Phaseolus vulgaris with the plant pathogen Colletotrichum species : A thesis submitted in partial fulfillment of the requirements for the Degree of Master at Lincoln University

Abstract

Colletotrichum gloeosporioides and C. acutatum are the main causal agents of mango anthracnose and also infect multiple other hosts in tropical and subtropical regions of the world. Common bean, Phaseolus vulgaris, being a cover crop, is commonly grown with mango plants in Uganda. These co-planted annual crop species can play a role as potential inoculum reservoirs in mango orchard cropping systems. The purpose of this study was to identify Colletotrichum species isolates obtained from naturally infected apple and avocado fruits using traditional and molecular means, to determine the pathogenicity of Colletotrichum species on common bean, Phaseolus vulgaris, an annual crop grown concurrently in mango cropping systems, and to assess the survival of Colletotrichum species on P. vulgaris crop debris under glasshouse conditions at Lincoln University. Five Colletotrichum species isolates were recovered from the fruit and identified based on morphological characteristics and molecular sequencing. Fungal characteristics including colony colour, conidia shape, and size, appressorial morphology, presence or absence of perithecia, and setae, and growth rates were used for species identification. Sequencing of the nuclear ribosomal internal transcribed spacer (ITS) region was conducted to confirm the identity of the Colletotrichum species isolates. Growth rates and conidial production of the isolates were assessed at 15, 20, 25, and 30 degrees Celsius in 12 hr light: 12 hr dark and constant dark. All the three isolates recovered from apple were identified as members of the C. gloeosporioides species complex and the two isolates recovered from avocado were identified as members of the C. acutatum species complex. For all isolates, the mean colony growth rate and conidial production were significantly higher (p=<0.05) when incubated under light: dark conditions than when under constant dark incubation. Temperature significantly affected (p=<0.001) the mean colony growth rate and spore production of the isolates, with the minimal growth rate and spore production of all isolates seen at 15 degrees Celsius and optimum between 25 and 30 degrees Celsius. To assess the pathogenicity of C. acutatum and C. gloeosporioides on P. vulgaris, common bean plants were inoculated by spraying with different conidial concentrations (10x2, 10x4, and 10x6 conidia/mL) of one isolate of C. acutatum and two isolates of C. gloeosporioides. Symptom development on leaves and pods was observed and recorded. All isolates were pathogenic on common bean, P. vulgaris, causing significantly (p=0.001) higher disease incidence and severity than the no pathogen controls, but there was no difference in incidence and severity resulting from the different inoculum concentrations. Colonies identified as the inoculating isolates were recovered from the lesions which developed on the bean plants confirming these as the causal agent. This is the first report of an isolate of the C. acutatum species complex as a potential pathogen of beans. To assess the ability of C. acutatum and C. gloeosporioides to survive on P. vulgaris crop debris, artificially inoculated bean plant material was buried in a potting mix: soil mixture at a depth of 5 cm or left on the surface for 6 weeks. The plant material was either coarsely cut or blended prior to inoculation with either C. acutatum or C. gloeosporioides conidial suspensions. Recovery of the C. acutatum and C. gloeosporioides from the bean plant tissue was assessed using dilution plating onto agar plates. In all instances, colonies morphologically identified as C. acutatum and C. gloeosporioides were recovered from the inoculated material incubated on the surface and buried at a depth of 5 cm after six weeks. However, recovery was significantly higher (p=0.001) from blended and buried plant material inoculated with either C. acutatum or C. gloeosporioides isolates than from the controls. Representative isolates recovered from the tissue were confirmed as members of the C. gloeosporioides species complex and the C. acutatum species complex by sequencing of the ITS gene region. Overall, this study has determined that isolates of the C. gloeosporioides species complex and of the C. acutatum species complex, known to be pathogens of mango, were also able to cause disease in common bean, a crop species commonly grown with mango in Uganda. These two species were also shown to survive for at least 6 weeks on inoculated bean crop debris and potentially act as a source of inoculum for mango in these cropping systems. For effective control of mango anthracnose disease, strict phytosanitary measures, including avoiding planting of alternative host crops or removal of the crop debris in a mango cropping system should be enforced. This will ensure the removal of potential sources of inoculum for new infections.