2.2.32
MODE OF SEED INFECTION AND MOLECULAR ANALYSIS OF MACROPHOMINA PHASEOLINA INFECTING MUNGBEAN

MJ FUHLBOHM1,2, EAB AITKEN1,2 and MJ RYLEY1,3

Cooperative Research Centre for Tropical PlantPathology, St Lucia, Brisbane, Australia; 2Department of Botany, University of Queensland 4072, Australia; 3Queensland Department of Primary Industries, PO Box 102 Toowoomba 4350, Australia

Background and objectives
The anamorphic fungus Macrophomina phaseolina [Tassi (Goid.)], is an economically important disease of mungbean causing rapid and premature death of plants either after flowering or under conditions of extreme stress. Infected stems turn ashy-grey. However, in mungbean, it is the seed-borne nature and seed transmission of the pathogen which is of most concern since, if infected seed is germinated, a wet, rapid-spreading, black rot develops on sprouts rendering them unfit for consumption [1]. It is not known how mungbean seeds become infected by this pathogen. In our initial studies we also found that even within individual plants, M. phaseolina is highly diverse when assessed with RAPDs, despite being an asexual fungus. These studies are being expanded to include detailed population studies with the screening of a larger number of fungal isolates from single field plants.

The aims of this study were to determine the possible modes of seed infection by M. phaseolina in mungbean and the infection process of pod and seed by histopathological techniques, and to determine the genotypic diversity within a small number of plants.

Materials and methods
Experiments designed to test the possibility of systemic or local establishment of M. phaseolina in seed, were carried out under controlled glasshouse conditions under a number of different water stress and temperature regimes. Direct pod inoculations using microsclerotia of the fungus were also performed. Sections of inoculated pods were prepared for histopathological staining and viewing in order to determine the minimum time required for the establishment of seed infection at 30°C. DNA was extracted from approximately 200 fungal isolates and screened across four primers previously characterized as revealing a high level of polymorphism.

Results and Conclusions

Despite the development of extensive symptoms of ashy stem blight on all plants after flowering, irrespective of the water stress/temperature regime, no seed infected with M. phaseolina was produced following inoculation methods remote from the pod. However, seed infection rates greater than 90% were obtained when pods were inoculated at all stages of pod development. The fungus was not detected in seeds from pods that developed after inoculation, even those on the same peduncle as inoculated pods. Histological staining revealed hyphae of M. phaseolina intercellular to the cells of the pod wall, but not in the seed 3 days after inoculation.

Infection of mungbean seed was found to be a result of direct pod penetration from localized inoculum. Similarly, in field observations, a sample of seed from semi-lodged mungbean plants with pods in direct contact with soil and soil splash on the pods was found to contain 20% internal infection of M. phaseolina. A sample from the non-lodged portion of the crop contained 2% infected seed. During other field samplings of mungbean crops, we sometimes found leaf spot caused by M. phaseolina. Koch’s postulates were satisfied under controlled conditions using an aqueous suspension of microsclerotia inoculated onto developing leaves [2]. This evidence strongly suggests that, in the field, leaf and seed infection is established by microsclerotia splashed onto leaves and developing pods, respectively, during rain. There is no evidence from these experiments that the fungus can establish itself systemically (leading to seed infection) in developing mungbean plants after inoculation of any plant parts other than pods.

Our previous studies of genotypic diversity of M. phaseolina using RAPDs demonstrated a high degree of differences even within a single plant. This is interesting result for an asexual fungus and warrants further investigation.

References
1. Lawn RJ, Imrie BC, 1991. Proceedings of the First Australian Mungbean Workshop pp 1-13.
2. Fuhlbohm MJ, Ryley MJ, Aitken EAB, 1996. Australasian Plant Pathology 25, 247-24.