The fungus Macrophomina phaseolina is the causal agent of several diseases of mungbean (Vigna radiata). One of these diseases, known as charcoal rot, causes spoilage of germinating seed lots, and occurs when seed contaminated with M. phaseolina is used for sprouting. The detection of the pathogen during seed testing prior to export leads to downgrading of the seed and a resultant financial penalty to the grain grower. The aims of this research were: to determine the location of M. phaseolina in diseased and symptomless tissue of mungbean plants; to determine the mode, site and timing of seed colonisation of mungbean; to determine the impact of biotic and abiotic factors on seed colonisation; to conduct an assessment of genotypic variation of M. phaseolina within single plants; to determine modes of transport of inoculum that contribute to
foliage infection and seed colonisation of mungbean; and to assess both pre- and post-harvest management strategies in order to reduce or prevent seed colonisation, or to minimise the process of seed transmission.
The location of M. phaseolina in mungbean plants was determined through serial sectioning of, and subsequent isolation from, naturally infected host tissue. A large proportion of the mungbean tissue infected by M. phaseolina was found to be symptomless. Moreover, there were large areas of ostensibly pathogen-free tissue separating infection foci, thus indicating a strong likelihood of independent aerial infections. A selection of 14 isolates obtained from serial sectioning were assessed for genotypic variation with six primers using RAPD analysis. Of the 36 bands that were scored, 78% were polymorphic and as a result, 12 distinct genotypes were detected. Polymorphisms were also detected amongst
isolates obtained from the same discrete infection area on single plants, which strongly suggests the occurrence of multiple aerial infections.
Various methods of controlled inoculation including soil infestation, pod and foliar inoculations, and artificial seed infestation, were used to determine how mungbean seeds are colonised by M. phaseolina. Additionally, most of these inoculation methods were coupled with a series of abiotic treatments (temperature regimes, watering regimes, application of herbicides) that were designed to initiate stress conditions within infected plants, and possibly trigger growth of M. phaseolina from the infection coiuts and colonise seed. Seed colonisation was established in vitro and in vivo when immature pods were directly inoculated with microsclerotia of M. phaseolina. At least two days exposure at
100% relative humidity (RH) was necessary to establish seed infection in detached mungbean pods that were inoculated with microsclerotia of M. phaseolina. Extensive seed infection was still obtained when one or more days of 100% RH was interrupted by up to three days at low humidity. All except one of the other methods of controlled inoculation failed to produce colonised seed even when combinations of stresses were applied. Only when the bipyridyliiun herbicide 'Spray Seed® 250' was applied to plants following the inoculation of mungbean stems within 13 cm of the pods, was seed colonised by M. phaseolina. This result raises the possibility that delayed harvesting of desiccated mungbean crops may promote further colonisation of mimgbean tissue, including seed.
Very strong evidence for the colonisation of mungbean seeds after deposition of soilsplashed
inoculum of M. phaseolina onto pods was obtained through field and laboratory-based studies. Soil-splashed inoculum (most likely microsclerotia) was also found to be the source of inoculum responsible for the development of Macrophomina leaf blight of mungbean at several regional sites. To further investigate this finding, areas of several mungbean crops growing in naturally infested soil were covered in hessian cloth to prevent soil-splash and assessments of the levels of seed colonisation between covered and uncovered areas were made. Although colonisation of seed in the covered areas was significantly lower than in the uncovered areas, covering the soil did not eliminate colonisation of seed. This result suggests that inoculum dispersal, leading to pod infection and subsequent seed colonisation, occurs not only in splashed-soil but also by other means. Soil transported to the extra-floral nectaries of mungbeans by ants was found to contain infective inoculum of
M. phaseolina. Furthermore, air-borne debris and dust collected in a trap contained viable microsclerotia of the pathogen. Isolates of M. phaseolina collected from both sources were pathogenic on mungbean seedlings, and suspensions of ant-transported soil and air-borne dust/debris infected mungbean pods and seed. This is the first report of both modes of inoculum dispersal. All three modes undoubtedly contribute to the total level of colonised seed, but their relative importance remains to be determined.
Several options for the management of charcoal rot in mungbean seeds were investigated. Application of the fungicide carbendazim to mungbean plants after flowering significantly decreased, but did not prevent, colonisation of mungbean seed by M. phaseolina. Consequently, this method of management holds little promise for mungbean growers. A large number of weeds common
in Australian mungbean fields were newly reported as hosts of M. phaseolina. Isolates obtained from the infected, but symptomless weeds were pathogenic on mungbean seedlings, thus indicating a lack of host-specificity toward mungbean. It is strongly suspected that the use of herbicides to control weeds in reduced and zero-tillage farming systems is increasing the risk of infection in subsequent mungbean crops through the build-up of inoculum in soil. This increased risk of infection may be further exacerbated by retaining stubble infested with M. phaseolina on the soil surface, thereby increasing the amount of inoculum that could be splashed onto plant organs.
Surface sterilisation, using sodium hypochlorite, significantly reduced colonisation levels in heavily colonised mungbean seed lines, but the process was not enhanced when a partial-vacuum was
introduced to the process. In some seed lots, up to 32% of the seed was colonised only on the seed coat (defined here as contamination), whereas the remainder was internally infected. Surface sterilisation also reduced the overall colonisation of 132 commercial lines by approximately one-third. Storage of seed for one month at either 4°C or 15°C significantly reduced colonisation levels in seed, whereas freezing treatments did not. Eradication of M. phaseolina from mungbean seed was possible through thermotherapy. However, the conditions required for eradication also contributed to large increases in abnormal germination levels and large losses in overall germination - an unacceptable trade-off for sprouters. A combination of thermotherapy and surface sterilisation of colonised mungbean seed may provide a more efficient process of seed treatment.