Infection of mungbean seed by Macrophomina phaseolina is more likely to result from localized pod infection than from systemic plant infection

Authors


Abstract

The ubiquitous fungal pathogen Macrophomina phaseolina is best known as causing charcoal rot and premature death when host plants are subject to post-flowering stress. Overseas reports of M. phaseolina causing a rapid rot during the sprouting of Australian mungbean seed resulted in an investigation of the possible modes of infection of seed. Isolations from serial portions of 10 mungbean plants naturally infected with the pathogen revealed that on most plants there were discrete portions of infected tissue separated by apparently healthy tissue. The results from these studies, together with molecular analysis of isolates collected from infected tissue on two of the plants, suggested that aerial infection of aboveground parts by different isolates is common. Inoculations of roots and aboveground parts of mungbean plants at nine temperature × soil moisture incubation combinations and of detached green pods strongly supported the concept that seed infection results from infection of pods by microsclerotia, rather than from hyphae growing systemically through the plant after root or stem infection. This proposal is reinforced by anecdotal evidence that high levels of seed infection are common when rainfall occurs during pod fill, and by the isolation of M. phaseolina from soil peds collected on pods of mungbean plants in the field. However, other experiments showed that when inoculum was placed within 130 mm of a green developing pod and a herbicide containing paraquat and diquat was sprayed on the inoculated plants, M. phaseolina was capable of some systemic growth from vegetative tissue into the pods and seeds.

Introduction

In Australia, the annual production of mungbean (Vigna radiata) and blackgram (Vigna mungo) varies considerably from year to year, mainly as a result of rainfall variability during the growing season and its use as an opportunist, dryland summer crop. Most of Australia's mungbean production is exported to overseas markets, particularly those of southeast Asia and the Indian subcontinent, with less than 5% of the annual production having the attributes required by the sprouting market. The need for consistent high quality has resulted in strict criteria, based on specific seed characteristics, being adopted by the Australian Mungbean Association (AMA; www.mungbean.org.au/grainqualitystandards.html).

In the early 1990s, a serious rot of sprouts from mungbean seed originating from Australia was reported in the USA and the UK when high temperatures (>28°C) were used in the sprouting process (Law & Law, 1991). Subsequent investigations revealed the causal agent to be Macrophomina phaseolina (Conde et al., 1991). By contrast, M. phaseolina has not been a major issue on mungbean sprouts in Japan, where producers use cooler water (<28°C) during sprouting (S. Donnelly, Blue Ribbon Seeds and Pulse Exporters, Kenmore, Queensland 4069, Australia, personal communication). As a result of the problems experienced in these overseas markets, in 1994 a test for charcoal rot was added to the AMA standards for export mungbean seed. Macrophomina phaseolina is widespread in the tropics and subtropics and is known to be pathogenic on at least 500 hosts of monocotyledonous and dicotyledonous angiosperms and of conifers (Dhingra & Sinclair, 1978). It is most commonly known as causing root and stem rots (variously referred to as charcoal rot, ashy stem blight and dry root rot) of summer field crops and vegetables (Dhingra & Sinclair, 1978). In Australia, M. phaseolina has been recorded on over 70 hosts (Australian Plant Pest Database; http://www.planthealthaustralia.com.au/go/phau/capacity-and-capability/information-support-systems/appd), including sorghum, soybean, navybean, mungbean, maize, sunflower and many common weeds (Fuhlbohm et al., 2012).

Microsclerotia are the primary inoculum of M. phaseolina in soil (Dhingra & Sinclair, 1978) and are either dispersed in soil or embedded in colonized plant tissues. Microsclerotia of M. phaseolina are released into the soil following host decay (Dhingra & Sinclair, 1978) or may be dislodged during tillage operations (Cook et al., 1973). Soil populations of microsclerotia were found to initially increase then later decrease as host debris was incorporated into the soil and decomposed (Short et al., 1980). Conidia that have oozed from pycnidia of M. phaseolina on plant parts have been implicated in the infection of Phaseolus vulgaris late in the growing season (Luttrell & Garren, 1952), but their exact role in the epidemiology of M. phaseolina on any host has not been elucidated.

The pathogen has been reported to be seed transmitted for many hosts (Richardson, 1979), including mungbean (Sharada & Shetty, 1987). Seed transmission of M. phaseolina can result in poor seed germination and rotting of seedlings (Gaikward & Sokhi, 1987) or the development of lesions on primary leaves and hypocotyls (Singh & Singh, 1982; Sharada & Shetty, 1987). Consequently, the use of Macrophomina-contaminated seed could lead to the introduction of this pathogen into new production areas (Sharada & Shetty, 1987).

Neergaard (1979) and Singh & Mathur (2004) describe several possible modes of infection of seeds: (i) direct infection from the mother plant via either vascular tissue (such as species of Fusarium and Verticillium that cause wilting of their hosts), or non-vascular tissues (such as smut and downy mildew pathogens and some fungal endophytes); (ii) indirect infection through the stigma and style (such as Claviceps spp., Didymella bryoniae, Ustilago spp.), the ovary and fruit wall (such as Ascochyta pisi, Phoma lingam, Phomopsis longicolla, Colletotrichum spp.), or the flower and fruit stalks (Colletotrichum lini, Septoria linicola); and (iii) from infected debris or soil adhering to the seed after threshing. These three modes of seed infection have been reported for M. phaseolina on different hosts. The work of Singh & Singh (1982), Sharada & Shetty (1987) and others support the concept of systemic growth of M. phaseolina from mother plants of sunflower, soybean and blackgram into the progeny seed. By contrast, other reports suggest that colonization of Phaseolus vulgaris by M. phaseolina may result from preharvest pod infection or infection from soilborne inoculum prior to germination (Songa & Hillocks, 1998). Establishment of M. phaseolina within seeds after pod/fruit inoculation has been described for green bean (Shama, 1991) and peanut (Jackson, 1965), while external contamination of seed by soil containing M. phaseolina was reported for harvested seed of kidney bean (Watanabe, 1972) and chickpea (Raabe, 1985).

Although there are many reports on the seedborne nature and seed transmission of M. phaseolina for different hosts, information on the mode of seed colonization is vague and unclear, and at times contradictory. Whilst it has been demonstrated that mungbean seed can be internally infected or externally contaminated by M. phaseolina (Rath & Routray, 1978; Sharada & Shetty, 1987), definitive studies investigating the mode or modes of mungbean seed colonization do not exist. Anecdotal evidence in Australia suggests that high levels of mungbean seed infection by M. phaseolina are usually associated with rainfall events during flowering and pod-fill. Furthermore, Fuhlbohm (2004) isolated M. phaseolina from soil peds, which had been splashed onto mungbean pods 400 mm above the soil surface after heavy rain. These findings and observations suggest that infection of the flowers and/or pods by M. phaseolina during or just after rainfall events results in significant levels of seed colonization.

Biotic and abiotic stresses, including water deficit, high temperatures after flowering and herbicides are known to affect the infection and invasion of hosts by M. phaseolina (Ghaffar & Erwin, 1969; Canaday et al., 1986; Smith & Wyllie, 1999). Herbicides, including those containing glyphosate and diquat, are used routinely in Australia to desiccate mungbean crops and promote even maturity prior to harvest (http://www.dpi.qld.gov.au/fieldcrops/8662.html). In the 1990s a small area of dead plants was observed in a crop of mungbean cv. Berken near Biloela (24·400729°S, 150·512838°E) in central Queensland. This area had been accidently sprayed with Spray.Seed®250 (135 g L−1 paraquat as paraquat dichloride and 115 g L−1 diquat as diquat dibromide, Syngenta Crop Protection Pty Ltd). Investigations revealed a high level (>80%) of seed colonization by M. phaseolina in non-sterilized seed obtained from the herbicide-affected portion of the crop but only 11% colonization in seed from the unaffected part of the crop (Fuhlbohm, 2004). This finding suggested that the herbicide spray had played a role in colonization of mungbean seed by M. phaseolina.

In this study experiments were conducted to determine the most likely mode of infection of mungbean seed by M. phaseolina. In turn, the findings were used to investigate possible management options, which are reported elsewhere. Three modes of infection were studied, namely (i) systemic invasion of the pathogen through mungbean plants after root infection by soilborne inoculum, (ii) systemic growth of hyphae into seeds from leaves and stems infected by airborne inoculum, and (iii) localized growth into seeds after infection of developing pods by airborne inoculum. Serial isolations from all parts of plants naturally infected in the field, assessments of genotypic variability of isolates from these isolations using RAPD analysis, and inoculation of seeds, roots, and aboveground vegetative and reproductive plant parts were conducted to obtain the necessary information. Nine combinations of different air temperatures and soil moisture levels were imposed on plants during some of the artificial inoculation studies. The role of herbicide application during pod formation was also investigated.

Materials and methods

Location of M. phaseolina in naturally infected mungbean plants

In 1996, 10 near-mature mungbean plants of cv. Black Pearl (designated Plant #1–Plant #10) were randomly selected from a commercial mungbean crop near Brookstead (27·754646°S, 151·448879°E), southern Queensland, dug from the ground with minimal disturbance to the root systems and placed in separate moistened plastic bags that were kept in a chilled container and transported to the University of Queensland, St Lucia. There the plants were refrigerated (4°C) until needed. Only three of these plants displayed symptoms characteristic of charcoal rot on mungbean, varying from small lesions on stems of plants #4 and #6 to a large contiguous lesion on the stem of plant #3. The remaining plants were symptomless.

Soil was removed from the root systems of the plants by rinsing in running tap water for 5 min. The plants were serially sectioned in 10 mm increments starting just below the plant–soil interface on the taproot, continuing up the stem and into the branches and peduncles. Leaves and petioles were not sampled. Depending on the size of the individual plant and the nature of lateral branching, up to 170 sections were assayed for M. phaseolina from each plant. Seed was removed from all mature pods that developed on the plants. After sectioning, the tissue pieces and seeds were surface sterilized in an aqueous solution of sodium hypochlorite (NaOCl) (2% available chlorine) for 3 min followed by a rinse in sterile distilled water (SDW), placed on 2% water agar (WA) amended with 0·1 g L−1 streptomycin sulphate and incubated in darkness at 30 ± 1°C for 4 days. Sections of plant from which colonies of M. phaseolina developed (identified by fluffy grey mycelium and small, black microsclerotia immersed in the agar) were counted as positive. This methodology is hereafter described as the ‘standard isolation and identification technique’, SIIT. Isolates were stored as colonized blocks of potato dextrose agar (PDA; Difco Laboratories) immersed in SDW or on PDA slopes immersed under sterile mineral oil in 20 mL McCartney bottles.

Genotypic diversity of M. phaseolina in naturally infected plants

Preparation of isolates for molecular analyses

Fourteen isolates of M. phaseolina were used in the RAPD genotypic analysis. Ten of the isolates were obtained from the sections of the main stems, peduncles and a single seed from plant #3 after serial sectioning, and the other four were obtained from peduncles and a single seed from plant #4 (Table 1). The pathogenicity of the 14 isolates was confirmed using the methodology described below in the seed inoculation section. The isolates were induced to sporulate using a modified method of Knox-Davies (1966). Circles of Whatman no. 1 filter paper (9 cm diameter; Whatman) were dipped in a 1:5 mixture by volume of ground raw peanuts and ether. After evaporation of the ether, the filter papers were autoclaved at 121°C and 100 kPa for 20 min then the papers were soaked in SDW, placed in sterile 90 mm diameter Petri dishes and inoculated with a 9 mm2 piece of colonized agar taken from the margin of a 4-day-old colony of M. phaseolina grown on PDA. The Petri dishes were sealed with Parafilm (Pechiney Plastic Packaging), incubated at 27°C and irradiated with a Philips TLD18W/08 lamp held 150 mm above the dishes. After 7 days the papers were re-soaked with SDW, left unwrapped for a further 5 days and rehydrated as required. After this time pycnidia formed on the entire upper surface of the filter papers. For some isolates, conidia oozed from the pycnidia and were retrieved from the surface of the filter paper using a sterile needle. For other isolates, the surfaces of the semidry papers were scraped with the edge of a glass slide to disrupt the pycnidia walls and expose the conidia. After retrieval the conidia were placed in SDW and distributed on the surface of PDA in a 90 mm diameter Petri dish. A conidium of each isolate of M. phaseolina was located using light microscopy and excised using a sterile 25-gauge hypodermic needle. The piece of excised agar with the single spore was placed on the surface of PDA in another Petri dish and incubated in darkness at 30 ± 1°C.

Table 1. Details of Macrophomina phaseolina isolates used in the study
UQ isolateBRIPaSource of isolatebLocalityUse
  1. a

    All UQ isolates apart from UQ 2100 were deposited in the Plant Pathology Herbarium (BRIP) of the Department of Agriculture, Fisheries and Forestry Queensland (DAFFQ), EcoSciences Precinct, Holland Park, Queensland, Australia.

  2. b

    All plants which had been serially sectioned (SS) were cv. Black Pearl.

2100?BiloelaFlower and pod inoc., herbicide study
332755165SS plant 4, branchBrooksteadRoot, foliar and seed inoculation
332855166SS plant 4, peduncleBrooksteadRoot, foliar and seed inoculation
332955167SS plant 4, peduncleBrooksteadRoot, foliar and seed inoculation
333055168cv. Berken, sproutsBiloelaDetached pod inoculations
335255169SS plant 3, main stem (soil level)BrooksteadRAPD analysis
336455178SS plant 3, main stemBrooksteadRAPD analysis
336655180SS plant 3, main stemBrooksteadRAPD analysis
336855181SS plant 3, main stemBrooksteadRAPD analysis
337555186SS plant 3, main stemBrooksteadHerbicide study
339055201SS plant 3, main stemBrooksteadRAPD analysis
339555206SS plant 3, peduncleBrooksteadRAPD analysis
341155219SS plant 3, peduncleBrooksteadRAPD analysis
341255220SS plant 3, peduncleBrooksteadRAPD analysis
343055234SS plant 3, peduncleBrooksteadRAPD analysis
345755257SS plant 4, peduncleBrooksteadRAPD analysis
346355263SS plant 4, peduncleBrooksteadRAPD analysis
347255272SS plant 4, peduncleBrooksteadRAPD analysis
348355283SS plant 4, seedBrooksteadRAPD analysis
350155286SS plant 3, seedBrooksteadRAPD analysis

Each single spore isolate was grown on V8 liquid medium that was prepared by adding 10·3 g of CaCO3 (Ajax Chemicals) to 750 mL V8 vegetable juice (Campbell's Soups). This solution was centrifuged at 1378 g for 20 min after which time the supernatant was diluted 1:4 with distilled water and autoclaved. Three small agar blocks of 4-day-old M. phaseolina growing on PDA were transferred to 40 mL liquid V8 medium and placed on an orbital shaker set at 140 rpm and 25°C. After 5 days, mycelia were collected by vacuum filtration onto Miracloth (Calbiochem-Novabiochem Corporation). The mycelia were stored in sterile plastic vials at −70°C.

RAPD analysis of isolates

Isolation of DNA

DNA was extracted from the harvested mycelia in duplicate by a modification of the method of Raeder & Broda (1985). After centrifugation for 1 h, the sample was incubated for 30 min with 5 μL of RNase (10 mg mL−1; Sigma Aldrich). The solution was extracted twice with one volume of chloroform. After overnight precipitation of DNA at −20°C, the sample was spun for 15 min. The DNA pellet was drained, washed twice with 500 μL of 70% alcohol and spun for 2 min, then the pellet was vacuum dried and resuspended in 50 μL of TE (10 mm Tris-HCl pH 8, 1 mm EDTA) buffer. The DNA concentration of each sample was measured using a Pharmacia Gene Quant RNA/DNA calculator (Pharmacia LKB Biochrom Ltd) and adjusted to a concentration of 25 ng μL−1 by adding Milli-Q plus 185 (MQ) filtered water (Millipore).

DNA amplification

PCR was carried out in 25 μL consisting of: 11·6 μL MQ water, 4 μL MgCl2 (25 mm) (Biotech International Ltd), 4 μL dNTPs (200 μm each of dATP, dCTP, dGTP and dTTP) (Biotech International Ltd), 2·5 μL 10 ×  buffer (Biotech International Ltd), 0·3 μm primer (Operon Technologies Inc.), 25 ng fungal DNA and 1·6 units Tth plus DNA polymerase (Biotech International Ltd). To ensure reproducibility of results, DNA from duplicate extractions of the same isolate was amplified. PCR was performed using a PTC-100 Programmable Thermal Controller (MJ Research, Inc.) with an initial DNA melt of 5 min at 94°C, followed by 39 cycles of: 1 min at 94°C, 1 min at 37°C, 2 min at 74°C; with a final 7 min extension step at 74°C.

Five 10mer primers were used in the comparison of the 14 isolates. The following primers were selected based on the results of an unpublished previous study: OPA-15 (5′-TTCCGAACCC-3′), OPH-04 (5′-GGAAGTCGCC-3′), OPH-05 (5′-AGTCGTCCCC-3′), OPH-08 (5′-GAAACACCCC-3′), OPH-09 (5′-TGTAGCTGGG-3′), OPP-02 (5′-TCGGCACGCA-3′).

After amplification, 5 μL loading dye (0·5% bromophenol blue, 0·5% xylene cyanole FF in 10 ×  TBE, pH 8·3) was added to each tube. Twenty microlitres of amplified DNA and loading dye was loaded on 1·5% agarose gels with 1 × TBE running buffer, run at 120 V until the bromophenol blue dye reached the bottom edge of the gel (c. 5 h), stained with ethidium bromide and photographed on a UV transilluminator (λ = 265 nm). All amplification band sizes were established by comparing them with a 100 bp molecular ladder.

The RAPD markers visualized on each gel were scored on the basis of the presence or absence of distinct fragments (bands). The presence of a marker in duplicate lanes of an isolate was scored as 1 and the absence as 0. The relationship between isolates was investigated using an unweighted pair group method with arithmetic means (UPMGA) tree derived from the combined data incorporating Jaccard's coefficient.

Root, aerial and seed inoculations

Inoculum preparation

A mixture of four single spore isolates was used for the root, aerial and seed inoculations. Three of the isolates (BRIP 55165, 55166, 55167) were obtained from aerial parts of the serially sectioned plant #4, as described previously, and the other (BRIP 55168) was obtained from diseased sprouts (Table 1). The pathogenicity of BRIP 55165, 55166, 55167 on mungbean seedlings was verified using the method described in the seed inoculation section below. Each of the four isolates of M. phaseolina was grown separately for 4 days at 30 ± 1°C in darkness in mungbean seed extract broth (MSEB) that had been prepared by boiling 100 g mungbean seed per L SDW for 15 min, straining the supernatant through three layers of cheesecloth, supplementing with 2% sucrose, then autoclaving at 121°C and 100 kPa for 15 min. The mycelia and microsclerotia that developed in MSEB broth were washed with SDW, collected by vacuum filtration onto Miracloth and press dried with paper towels to remove excess moisture.

Soil preparation

A potting mixture of washed medium-coarse sand, peat and red ferrosol soil (1:1:3) was sterilized for 2 h using aerated steam (100°C) and, after cooling, the presence or absence of M. phaseolina was determined by a method modified from Mihail & Alcorn (1982). Tenfold serial dilutions of a 1 in 10 solution of wet soil and water were made and 10 mL of each dilution was added to 90 mL molten cooled WA containing pentachloronitrobenzene (1 g L−1) and streptomycin sulphate (0·1 g L−1) (MP medium). Each soil dilution/agar mixture was dispensed in equal volumes (20 mL) between five 90 mm diameter Petri dishes, incubated at 30°C in the dark for 4 days and assessed for the presence of M. phaseolina. The pathogen was not detected in any of the dilutions.

The field capacity (FC) of the soil mix was determined to be 32·8% after thorough wetting of the soil followed by drainage on a flat surface for 48 h, then measuring the moisture content (MC) of the soil sample. Four replicates were used in this determination.

Soil MC was determined by the following formula:

display math

where W1 = weight of soil (g) prior to drying, and W2 = weight of soil (g) after drying at 60°C for 72 h.

Plant growth conditions

The mungbean cv. Berken was used in root, aerial and seed inoculation experiments. Planting seed was sourced from plants that had been grown in sterile potting mix under optimum growing conditions in a glasshouse. A random sample of 20 seeds was plated directly onto PDA plates and incubated at 28°C for 7 days, but colonies typical of M. phaseolina did not develop from any seed; the seed was therefore considered to be ‘M. phaseolina-free’. Plants were grown in new 200 mm diameter pots lined with a plastic bag, containing 3·466 kg of the soil mixture which had been dried to 0% MC in ovens at 60°C for 3 days. The surface of the soil was covered in a layer of high-density small white plastic beads (Kemcor Australia Pty Ltd) to reduce evaporation. Four pots (replicates) each with three plants were used for each combination of inoculation method, temperature and watering regime.

Root inoculations

The potting mix was artificially infested with M. phaseolina by thoroughly mixing 0·5 g of press-dried inoculum, containing equal proportions of the four isolates, per 1 kg of sterilized mix which was then placed in the pots as described above. After the mix was watered to FC, 10 Macrophomina-free seeds were sown in each pot, which were then placed in one of the nine temperature (designated as high T, medium T and low T) × soil moisture (designated as high M, medium M and low M) combinations described below. After 35 days, 30 plants randomly selected from each temperature regime and grown under the high M regime were assayed for the presence of M. phaseolina in the roots by the SIIT method, and the remaining plants were thinned to three per pot. The experiment was repeated except that only the low and medium temperature regimes were used in combination with the three moisture regimes, because in the first experiment all plants in the high temperature regime died before maturity, irrespective of the moisture regime.

Pre-flowering aerial inoculations

Macrophomina-free mungbean seeds were sown in pots containing sterilized potting mix and maintained at each of the respective temperature regimes under the high M regime. Two weeks after sowing, the plants were thinned to three per pot, and 2 weeks later the aerial parts of the plant were sprayed to run-off with a suspension consisting of a mixture of equal proportions of the four isolates (total 0·25 g) in 20 mL SDW. At this stage the plants had not flowered. After inoculation, plants were sealed in pre-moistened plastic bags and incubated at 30 ± 1°C for 3 days. The plants were then returned to their original temperature regime and subjected to one of the three watering regimes for the remainder of the experiment.

Serial sections were made from three mature randomly selected plants in each of the medium T × high M and medium T × medium M regimes, 43 days after inoculation. A 10 mm long segment of stem was excised every 50 mm along the stem from ground level, and was assayed for the presence M. phaseolina by the SIIT. Sections of petioles, peduncles and pedicels from these plants were also assayed, and pods from the six plants were removed for seed testing. The aerial inoculations were conducted a second time, with the exceptions described above for the root inoculations.

Seed inoculation

Macrophomina-free seeds were placed in a mixed isolate inoculum suspension described in the aerial inoculation section for 80 min at 22°C under diffused artificial light. The seeds were then planted in trays containing moist sterile vermiculite, enclosed in a pre-moistened plastic bag and incubated for 48 h at 25°C in the dark. When they had germinated, the temperature was increased to 28°C for a further 48 h. Seedlings with discrete cotyledon or hypocotyl lesions were carefully removed from the vermiculite and transplanted into pots containing sterilized soil, with up to 20 seedlings per pot. Seedlings with severe hypocotyl rotting were not used. The pots were moved to a temperature-controlled glasshouse facility maintained at 30 ± 2°C and covered in a pre-moistened plastic bag while the seedlings established new roots and root hairs after transplant damage. The bags were partly removed over a period of 1 week to allow progressive introduction to the drier atmosphere, then survivors were thinned to three per pot and exposed to one of the nine T × M regimes. In the second experiment, results for the medium T regime were not obtained due to a severe mite infestation that defoliated the plants prior to pod formation.

Post-inoculation temperature × soil moisture regimes

After the respective inoculation methods outlined above were completed, the pots of plants or seeds were transferred to one of three controlled-temperature glasshouse facilities, in which the temperature regimes are described as low T (mean minimum and maximum temperatures of 21·3 ± 1·1°C and 26·6 ± 2·2°C respectively), medium T (29·9 ± 0·6°C and 31·8 ± 1·0°C respectively), and high T (33·2 ± 0·8°C and 35·9 ± 2·3°C respectively). At the start of each experiment and inoculation time, the soil mixture was initially watered to field capacity (FC) but at the initiation of the experiments the pots were subjected to one of three watering regimes until the completion of the experiments. All regimes were based on a weight for weight basis. In the high water regime (high M), water was added to pots every day to ensure that the soil was maintained at FC. In the medium M soil moisture regime, soil moisture fluctuated between wilting point (WP) and FC. The soil was allowed to dry and at the first sign of plant wilting, water was added to increase the soil moisture content up to FC, a process that was repeated for the duration of the experiment. This weight of water is the plant available water capacity (PAWC) of that soil. The low M soil moisture regime consisted of fluctuations between WP and a soil moisture content of 20% by adding a weight of water equivalent to 30% PAWC for the duration of the experiments.

Over the course of the experiments, the weight of dry matter added by the growing plants was considered to have an insignificant effect on the predetermined FC value. The plants were subject to glasshouse diurnal light conditions (12 h 20 min–13 h 50 min daylight) during the experiment.

Control treatment

Control checks for all inoculation methods were established by soaking Macrophomina-free seed in SDW for 80 min followed by planting in M. phaseolina-free soil at high M. The pots were then incubated at one of the nine T × M regimes. After 4 weeks the young plants were thinned to three per pot, sprayed with SDW until run-off, sealed in pre-moistened plastic bags and incubated at 30 ± 1°C for 3 days. The plants were then returned to their particular temperature × moisture regime for the remainder of the experiment. Nine plants (three pots of three plants) served as controls for each treatment. The root system from one plant in each of the three pots of plants grown at the medium temperature regime was assayed for M. phaseolina at maturity by the SIIT.

Seed harvesting and testing for M. phaseolina

Mungbean plants have an indeterminate growth and flowering habit, so mature pods (black with dry, brittle walls) were collected progressively from all test plants. For individual plants, seed was extracted from pods and combined for the M. phaseolina detection assay irrespective of when they were collected. The seeds were placed equidistantly inside clean and sealable plastic containers on a sheet of sterile germination paper (Ekwip), pre-wet to saturation with SDW, and covered with wet facial tissues (Kleenex). Excess water was removed and the plastic containers were sealed and placed inside a dark incubator for 3 days at 30 ± 1°C. During incubation the germination papers were rewet periodically with SDW to maintain a high humidity. Mungbean seeds and sprouts were then visually assessed for signs and symptoms of infection by M. phaseolina (Fig. 1a).

Figure 1.

Symptoms and signs of infection on mungbean after inoculation with Macrophomina phaseolina on (a) sprouts; (b) leaves after pre-flowering aerial inoculation; (c) stem after pre-flowering aerial inoculation – note red-brown stem lesions; (d) pod with embedded microsclerotia and pycnidia after inoculation with a M. phaseolina-colonized agar plug on a peduncle <130 mm from the pod, followed by an application of the herbicide Spray.Seed®250 7 days later.

Flower and pod inoculations

Three experiments involving inoculations at different flowering and pod stages were conducted. In all experiments, surface sterilized Macrophomina-free seeds of cv. Berken were planted in sterile potting mix in 150 mm diameter pots in a glasshouse and watered to FC. After 7 days the plants were thinned to one per pot.

In the first experiment, inoculations were performed at the late-flowering stage (approximately 45 days after sowing, DAS) when pods of different ages and some flowers coexisted on single racemes. Ten plants were inoculated with M. phaseolina, and four plants that were sprayed with SDW only served as controls. In the second experiment, plants were inoculated at either 36 DAS when there were flowers and young pods <20 mm long, or 43 DAS after sowing when there were flowers and pods up to 60 mm long. At each stage 10 plants were inoculated with M. phaseolina, and another 10 plants were sprayed with SDW (control plants). In the third experiment plants were inoculated at one of the following six stages: 35 DAS with only flowers (flowering, F), 41 DAS with pods <30 mm long (early pod fill, P1), 45 DAS with pods >30 mm and <60 mm (mid pod fill, P2), 50 DAS with pods >60 mm but <100 mm (mid–late pod fill, P3), 55 DAS with pods >100 mm (late pod fill, P4), and 65 DAS with full expanded and young pods (S). Ten plants were inoculated with M. phaseolina at each of the developmental stages and 10 other plants sprayed with SDW at late pod fill served as controls.

In all three experiments, flowers and pods were inoculated by spraying a microsclerotial suspension of M. phaseolina isolate UQ 2100, at 0·125 g microsclerotia per 20 mL SDW, until run-off. Microsclerotial inoculum was prepared by growing the isolate on MSEB for 10 days at 30°C in the dark, then washing and homogenizing the colonies in SDW. The homogenate was dried in an incubator at 40°C for 24 h, and the microsclerotia ground in a mortar and pestle and screened through a sieve with 175 μm mesh. After inoculation, the pots and plants were sealed in pre-moistened plastic bags and incubated in a temperature-controlled glasshouse at 28 ± 2°C for 72 h in natural light (12 h 20 m–13 h 50 m daylight). The pots were then returned to another glasshouse (mean minimum temperature 24·5 ± 1·1°C, mean maximum temperature 33·2 ± 3·1°C) for the duration of the experiments.

Seeds were collected at plant maturity in all experiments and assayed for the presence of M. phaseolina by the method described above, except that they were not surface sterilized. In the second experiment, information on the source of the seed (from symptomless pods or pods displaying symptoms of infection) was also obtained. Seed from pods that developed on plants subsequent to the inoculation events in the third experiment were also harvested and assayed for M. phaseolina.

Detached pod inoculations

Green pods at late pod fill (P4) were harvested from plants of cv. Berken that were grown from M. phaseolina-free seed in sterilized soil under glasshouse conditions. After collection, the pods were immersed in 100% ethanol for 10 s, allowed to dry and then placed for 30 min in a suspension of 0·125 g microsclerotia per 20 mL SDW (equal proportions of isolates BRIP 55165, 55166, 55167 and 55168) in a glass Petri dish. The inoculated pods were then placed on inverted sterile plastic test tube racks inside plastic storage boxes, the bottom of which were lined with sheets of germination paper (Ekwip).

Pods were subjected to different sequences of days in the dark at high or low relative humidity (RH) in an incubator maintained at 32 ± 1·3°C. High RH was obtained by saturating the germination paper with water and sealing the plastic boxes with lids. The presence of free water on the blotter surface ensured that the RH was at or very near 100%. Low relative humidity was achieved by using dry germination paper, not sealing the plastic containers, and exposing them to the 31 ± 3·6% (low) ambient RH of the incubator. The temperature and RH of the incubator was constantly monitored with a thermohydrograph (Hanna Instruments, model HI8564). Different humidity regimes were established by transferring individual pods between low and high RH plastic containers for various periods over the course of the experiment. Each humidity regime treatment consisted of eight replicates of single pods placed in different plastic boxes. Pods soaked in sterile water for 30 min and placed similarly in the plastic racks served as controls. Low and high RH plastic containers were randomly placed in the incubator.

After the prescribed incubation period, pods were removed from each of the sealed plastic containers, the seeds extracted, combined and washed in a solution of NaOCl (2% available chlorine) for 60 s followed by a rinse in SDW. The surface sterilized seeds were placed on 2% water agar amended with 0·1 g L−1 streptomycin sulphate, incubated for 3 days at 31 ± 1°C, and examined for the presence of M. phaseolina colonies. Between 64 and 93 seeds from the test and control-inoculated pods were assayed for M. phaseolina.

The impact of desiccant herbicides

Inoculation of vegetative tissue

Mungbean plants of cv. Berken raised from M. phaseolina-free seed were grown individually in sterile soil mix in 150 mm pots. The plants were grown in a glasshouse at 25–34°C until mid-pod fill (c. 45 days DAS) and were watered regularly to FC to ensure they were not moisture stressed. At this stage the pods were green and 30–60 mm long. Plants were then inoculated by one of the methods outlined below.

Aerial inoculation

Microsclerotial inoculum of M. phaseolina isolate BRIP 55186 was prepared using the same methodology described in the previous section, and a suspension of this isolate was sprayed onto the vegetative parts of nine plants at 0·250 g microsclerotia per 20 mL water. Another three plants were inoculated with SDW only (control treatment). The flowers and pods of all plants were covered with small clip-lock plastic bags prior to inoculation and for a further 3 days after inoculation. Inoculated plants were incubated in a glasshouse for 3 days at 30 ± 1°C then returned to a 25–34°C glasshouse for a further 4 days. Six of the nine plants inoculated with M. phaseolina and the three plants inoculated with SDW were then sprayed until run-off with Spray.Seed®250 at 20 mL product per litre SDW. The remaining plants were not subjected to the herbicide application. Pods were harvested from all plants 5 days later and the non-surface sterilized seeds were assayed for M. phaseolina using the sprouting method described above. A seed was considered colonized if signs of infection were evident on ungerminated seed or on sprouts.

Toothpick inoculation

Sterile wooden toothpicks were placed on an actively growing culture of M. phaseolina (isolate UQ 2100) grown on PDA at 30 ± 1°C for 5 days at which time they were covered with microsclerotia and sparse mycelium. Macrophomina-infested toothpicks were inserted at the cotyledonary scar of four plants, located 30–60 mm above the soil line, the wounding sites being 530–680 mm from the pods. Non-inoculated, sterile toothpicks were inserted at the same site on four other plants.

The inoculation site and the immediately adjacent stem were wrapped in Parafilm for 3 days and transferred to a temperature-controlled glasshouse maintained at 30 ± 1°C. All plants were then moved back into the 25–34°C glasshouse and, after 4 days, Spray.Seed®250 was applied until run-off using a hand atomizer at a concentration of 20 mL product per litre SDW. Three non-inoculated and three non-wounded plants were sprayed at the same time and incubated under the same conditions as inoculated and herbicide-sprayed plants.

Colonized agar plug inoculations

Two separate experiments were conducted, one incorporating Spray.Seed®250 application and the other using the herbicides Glyphosate360® (360 g L−1 glyphosate as the isopropylamine salt, Nufarm Ltd) and Reglone® (200 g L−1 diquat as diquat dibromide, Syngenta Crop Protection Pty Ltd). For both experiments isolate UQ 2100 was grown on PDA for 5 days at 30 ± 1°C, after which 4 mm diameter plugs were cut from the colonies. The plugs were placed on the surface of stems at the nodes or on peduncles at distances varying from 20 to 530 mm from immature pods on 44 plants to be sprayed with Spray.Seed®250, or between 20 and 100 mm from the pods for the 26 plants to be sprayed with either Glyphosate360® or Reglone®. Control treatments for both experiments consisted of plants inoculated with uncolonized PDA plugs applied at distances of 10–150 mm from the pods prior to herbicide application (eight plants per herbicide), and nine plants inoculated with M. phaseolina at distances of 30–120 mm from the pods but not sprayed with herbicide.

The inoculation site and immediately adjacent stem were covered in Parafilm and plants were transferred to a temperature-controlled glasshouse maintained at 30 ± 1°C for 3 days. All plants were subsequently moved back into the 25–34°C glasshouse for 4 days then sprayed with Spray.Seed®250 at a rate of 20 mL product per litre SDW, Glyphosate360® at 10 mL product per litre SDW, or Reglone® at 20 mL product per litre SDW until run-off using hand atomizers.

At the conclusion of both experiments, pods were removed from the plants 7 days after herbicide application and the seeds extracted. All seed harvested from each pod was divided into two equal lots, one of which was surface sterilized as described above and the other not sterilized. Seeds were counted as positive for the presence of M. phaseolina when there was grey mycelium and black microsclerotia on diseased seeds and charcoal rot symptoms on affected sprouts.

Soil infestation, pre-flowering aerial inoculation and artificial seed colonization

The methods used to grow plants prior to inoculation, produce inoculum (mixture of M. phaseolina isolates BRIP 55165, 55166, 55167 and 55168) and inoculate plants using the soil infestation, pre-flowering aerial inoculation and artificial seed colonization techniques were identical to those described above, except that after inoculation the plants were incubated at only one T × M regime. For each treatment, four replicate pots, each containing three plants, were used. There were three control treatments, consisting of inoculation with M. phaseolina by each of the three methods and not sprayed with herbicide, and no inoculation with and without herbicide application. After inoculation by one of the three methods, all pots (including those of the control treatments) were transferred to a controlled-temperature facility maintained at mean minimum and maximum temperatures of 29·9 ± 0·6°C and 31·8 ± 1°C respectively. The pots were arranged randomly and the soil moisture was maintained at FC for the duration of the experiment. When the plants had reached mid- to late pod fill stage, Spray.Seed®250 was applied using the same method described above. The seeds were not sterilized.

Seed harvesting and testing for M. phaseolina

For the toothpick and colonized agar plug inoculation techniques, pods were removed from the plants 7 days after herbicide application and the seed from each pod was divided into two lots, one of which was surface sterilized using the methods described above and the other left unsterilized before incubating on sterile paper in trays, as described above. For the other inoculation methods, pods were collected progressively when mature and the seed extracted but not sterilized before assaying for M. phaseolina.

A summary of all of the above inoculation methods is provided in Table 2.

Table 2. Summary of inoculation methods and conditions used in a study of mungbean seed infection by Macrophomina phaseolina
Inoculation typeInoculumInoculation methodPre-inoculation conditionsInoculation conditionsPost-inoculation conditions
  1. a

    Mixture of equal weights of inoculum of BRIP 55165, 55166, 55167 and 55168.

  2. b

    Low, medium or high moisture regimes × low, medium or high temperature regimes; details in Materials and Methods.

  3. c

    Spray.Seed®250 (135 g L−1 paraquat as paraquat dichloride and 115 g L−1 diquat as diquat dibromide).

  4. d

    Glyphosate360® (360 g L−1 glyphosate as the isopropylamine salt), Reglone® (200 g L−1 diquat as diquat dibromide).

RootMixaMixed in potting mixOne of nine T × M combinationsbOne of nine T × M combinations
Pre-flowering aerialMixSprayed mycelia suspensionHigh M  ×  low, medium, high T30°C for 3 days, then one of nine T × M combinations
SeedMixImmersed in mycelia suspension80 min at 22°C48 h at 25°C, 48 h at 28°C, 7 days at 30°C, one of nine T × M combinations
Flower and podUQ 2100Sprayed microsclerotia suspension25–34°C72 h at 28°C, 24·5–33·2°C until plant maturity
Detached podMixSprayed mycelia suspension25–34°C32°C, sequences of low and high humidity
Aerial and pod + herbicideBRIP 55186Sprayed mycelia suspension25–34°C3 days at 30°C, 4 days at 25–34°C then sprayed with Spray.Seedc
Toothpick + herbicideUQ 2100Colonized toothpick25–34°C3 days at 30°C, 4 days at 25–34°C then sprayed with Spray.Seed
Agar block + herbicideUQ 2100Colonized agar plug25–34°C3 days at 30°C, 4 days at 25–34°C then sprayed with Spray.Seed, Glyphosated or Regloned
Root + herbicideMixMixed in potting mix29·9–31·8°C then sprayed with Spray.Seed
Pre-flowering aerial + herbicideMixSprayed mycelia suspension25–34°C29·9–31·8°C then sprayed with Spray.Seed
Seed + herbicideMixImmersed in mycelia suspension80 min at 22°C29·9–31·8°C then sprayed with Spray.Seed

Results

Location of M. phaseolina isolates from dissected mungbean plants

Macrophomina phaseolina was always isolated from sections of stems and branches that displayed symptoms of charcoal rot and occasionally from symptomless plant sections of all 10 plants which were serially sectioned. Figure 2 displays the results of the serial sectioning for plants #3, #4 and #7. Plant #3 was the only one of the 10 dissected plants that had the entire length of the main stem infected by M. phaseolina. However, on some branches of this plant there were sections of infected tissue, including pods that were separated from other infected areas by apparently healthy tissue. In other plants, single or multiple 10 mm lengths of Macrophomina-infected tissue on stems, branches and peduncles were separated by healthy tissue from which M. phaseolina was not isolated. Infected seed was obtained from six of the 10 plants, and for 68% of this seed, M. phaseolina was not found in the subtending peduncle tissue or in stem tissue at the base of peduncles.

Figure 2.

Pictorial representations of three mungbean plants naturally infected with Macrophomina phaseolina. Pods are depicted as elongated oval shapes clustered at the ends of peduncles. Thickened areas on the stems, branches, peduncles and pods represent the location of M. phaseolina as determined by isolation from tissue segments. BRIP numbers and arrows signify the locations from which isolates used in the RAPD analysis were collected.

Genotypic diversity of M. phaseolina in naturally infected plants

All of the primers provided reproducible RAPD patterns. A total of 36 bands were scored, but faint or ambiguous bands were not scored. Twenty-two percent of the amplified bands were common to all isolates, while 78% were polymorphic. Fourteen percent of the bands were unique to a single isolate. There was no clustering of grouped plant isolates (Fig. 3). In addition, these data reveal polymorphism between isolates obtained from the same discrete infection area. For example, isolates BRIP 55219 and BRIP 55220, obtained from consecutive peduncle segments on plant #3, were not closely related. Two groups of clonal isolates, (BRIP 55201 and BRIP 55206) and (BRIP 55180 and BRIP 55181), from the main stem of plant #3 were apparently evident from the data. Each member isolate of the two clonal pairs was obtained from a maximum separation distance of 25 mm on the plant.

Figure 3.

Dendrogram constructed with UPGMA clustering method among 14 isolates of Macrophomina phaseolina. Similarities were computed from 36 RAPD loci. The scale in the dendrogram is the genetic similarity coefficient calculated according to Dice (1945).

Root, aerial and seed inoculations

Uninoculated plants reached maturity and produced seed 60–120 days after sowing, depending on the temperature and watering regime imposed. All flowers on plants exposed to the high T regime, irrespective of the M regime and whether they were inoculated or not, aborted with the result that no pods or seeds developed.

Root inoculations

Macrophomina phaseolina was successfully isolated from 60, 83 and 97% (= 30) of 35 day-old root systems grown at field capacity from the low, medium and high T regimes respectively. Charcoal rot symptoms did not develop on plants grown at the low T regime for any M regime. Dark, red-brown lesions typical of those caused by M. phaseolina developed near the soil level on surviving plants grown at the medium T, and on some of these plants the lesions expanded rapidly and turned light grey, with the plants dying before maturity. At the medium and high T regimes the symptoms were more severe in plants subjected to water stress (medium M and low M). Every plant grown at high T developed a basal stem lesion that developed rapidly and turned light grey and they all died before or at flowering. Macrophomina phaseolina was not isolated from seed that developed at any T × M combination, irrespective of whether or not they were inoculated.

Pre-flowering aerial inoculations

Circular irregular, red-brown lesions 2–5 mm in diameter developed on leaves and stems of plants in all T × M regimes within 3 days of inoculation (Fig. 1b,c). At low T, the lesions did not expand at any time and plants remained alive for as long as uninoculated plants, irrespective of the watering regime. At medium T, lesions on plants in the high M remained discrete until pod fill, then expanded rapidly, killing plants prior to the uninoculated plants reaching maturity. By contrast, in the other two M regimes at this temperature, although lesions developed on inoculated plants before pod fill, the plants were not killed prematurely. At high T, the lesions on inoculated plants developed rapidly and most plants died before reaching maturity. Every flower on inoculated plants subject to high T aborted. As the stem lesions expanded they became pale and dry, with pycnidia and microsclerotia developing in or on the affected tissue.

Partial-serial sectioning at maturity on aerially inoculated plants grown at medium T revealed the presence of M. phaseolina throughout much of the stem tissue. In some cases, such as plants #2, #3 and #4, there were large and apparently contiguous regions of M. phaseolina infection (Fig. 4). Macrophomina phaseolina was not only isolated from diseased tissue, but also from symptomless tissue. As for the root inoculations, M. phaseolina was not isolated from seed of either inoculated or uninoculated plants at any T × M combination.

Figure 4.

Location of Macrophomina phaseolina in mungbean plants whose aboveground parts had been inoculated at the pre-flowering stage, as determined by isolations from partial serial sections of stems and pedicels.

Artificial seed infestation

Most transplanted seedlings died within 7 days of inoculation, but lesion and disease development on the survivors was similar to that described for the aerial inoculated plants at the same T × M combination. Lesions on inoculated plants from low T did not expand irrespective of the watering regime. For plants grown at high T, every flower aborted on these plants with the consequence that no pods developed. In the medium T regime, lesions expanded rapidly and most plants remained alive until maturity.

No seed was produced on root-inoculated and aerial-inoculated plants or on plants that grew from inoculated seed developed symptoms of charcoal rot when sprouted, irrespective of the T × M combination. Symptoms of charcoal rot infection were not observed on uninoculated plants or on sprouts that developed from seeds of uninoculated plants.

Flower and pod inoculations

In all three experiments, most inoculated flowers aborted and many young pods (P1 and P2 stages) abscised from the plants after inoculation, but older pods were less prone to abscission. This phenomenon did not occur on plants inoculated with water only. Many of the pods inoculated with M. phaseolina developed small galls composed of proliferated host tissue, which gradually turned red-brown. However, by pod maturity the galls were no longer evident and appeared as white specks on the surface of the pods. Later, microsclerotia and pycnidia developed on the infected pods.

In all experiments, no seed harvested from inoculated pods exhibited any external signs or symptoms of infection by M. phaseolina prior to incubation on the germination trays. In experiment 1, 34% of seeds were colonized following inoculations at the late flowering stage (when flowers and pods of various sizes were present; Table 3). In experiment 2, >90% of seeds from pods with lesions were infected, irrespective of the pod size at the time of inoculation, whereas <12% of seeds from lesion-free pods were infected. In experiment 3, 13% of seed from plants inoculated at flowering were infected, while 74–93% of seeds from inoculated pods were infected, irrespective of the size of pods at inoculation. No infected seeds were obtained from pods that developed on the same raceme as earlier inoculated pods, even if infected seeds had been obtained from those pods. In all three experiments, none of the seed collected from plants inoculated with SDW produced charcoal rot symptoms on developing sprouts or rotted seed.

Table 3. The effect of inoculation of Macrophomina phaseolina at different flowering and podding stages on the germination and colonization of mungbean seed
Experiment and stage of development at inoculationNo. plantsSeed characteristics
No. seeds% germinated% diseaseda
  1. a

    Total % of Macrophomina-infected seedlings including non-germinated (damped-off) seeds and diseased seedlings.

Experiment 1
Late flowering10100n/a34
Not inoculated473n/a0
Experiment 2
Flowers, and pods <20 mm    
Pods with symptoms10714·3100
Pods without symptoms1019394·311·4
Not inoculated10100n/a0
Flowers, and pods <60 mm
Pods with symptoms106328·693·7
Pods without symptoms1013797·38
Not inoculated10100n/a0
Experiment 3
Flowering1015010013
Pods <30 mm (P1)10999592
Pods >30 mm, <60 mm (P2)101009479
Pods >60 mm, <100 mm (P3)101509393
Pods >100 mm, but not mature (P4)101007274
Pods mature (S)10300980
Not inoculated (P4)101001000

Detached pod inoculations

At least two, but not necessarily consecutive, days of 100% RH were necessary to establish seed infection from inoculated pods at 32 ± 1·3°C (Table 4). High levels of seed infection were obtained when one or more days of 100% RH was interrupted by up to 3 days at low RH. None of the constant low RH regimes (2–5 consecutive days) led to the establishment of seed infection. Seeds that developed in pods inoculated with water only were not infected with M. phaseolina.

Table 4. Effect of different sequences of exposure of excised mungbean pods to high and low relative humidity (RH) on seed infection by Macrophomina phaseolina
Programme of days at high (●) or low RH (○)aInfected seed (%)
123456789
  1. a

    High RH = 100%; low RH = 31 ± 3·6%.

        0
       27
      53
      61
     85
    51
    67
     63
    69
   88
  72
    80
   72
  80
95
       0
      0
     0
    0

Herbicide interactions

Plants whose aboveground parts had been inoculated with an aqueous suspension of M. phaseolina inoculum and subjected to an application of Spray.Seed®250 produced a total of 94 seeds, 85 of which (90%) were colonized by M. phaseolina. Microsclerotia and pycnidia developed on the surface of pods from which infected seeds were collected (Fig. 1d). The pathogen was not isolated from the 64 seeds produced on plants inoculated but not sprayed with the herbicide or from the 52 seeds produced on plants which had not been inoculated but which were sprayed with Spray.Seed®250.

Macrophomina phaseolina was not obtained from any seed produced on plants inoculated with infested toothpicks regardless of whether or not the plants were then subjected to Spray.Seed®250 application. By comparison, Macrophomina-colonized seed developed on 20 of the 34 plants which had colonized plugs placed within 130 mm of the pods prior to spraying with Spray.Seed®250, and of the 20, 17 had plugs placed within 50 mm of the pods. A total of 281 seeds were collected from these 20 plants, and 62 of 142 seeds (44%) that were not surface sterilized were colonized by M. phaseolina, while 33 of the 139 surface-sterilized seed (24%) were infected with M. phaseolina. None of the seeds that developed on plants that had been inoculated with Macrophomina-colonized agar plugs at distances >130 mm from the pods were infected with the pathogen.

No seed produced on plants that were subject to root, aerial or seed inoculation before being sprayed with Roundup360® or Reglone® or on control plants were colonized by M. phaseolina.

Discussion

These investigations conclusively demonstrated that seeds of mungbean became infected by M. phaseolina after inoculum of the pathogen was sprayed onto the surfaces of developing green pods, irrespective of the age of pods and whether they were detached or attached to plants. Inoculation of roots and of aboveground plant parts prior to flowering by inoculum of M. phaseolina under several combinations of moisture and temperature regimes did not result in the infection of mungbean seed. Serial sectioning of naturally and artificially infected plants and molecular analysis of isolates obtained from stems of the former revealed that isolates obtained from multiple sites on the same plant represent distinct genotypes. These findings suggest that infection of aboveground plant parts of mungbean plants results from multiple infections by different isolates of M. phaseolina, rather than from systemic infection. Furthermore, pods that developed on the same raceme subsequent to inoculated pods were not infected, demonstrating that the initial pod infections remained localized and did not spread into other pods located on the same raceme.

These findings are supported by the results of Hill et al. (1981) who serially sectioned portions of soybean stems following inoculation with conidia of Phomopsis sojae and found that the infections remained localized and spread very little from the initial point of infection. They concluded that the sudden appearance of pycnidia on soybean plants late in the season probably resulted from multiple, localized infections rather than systemic infection as suggested by Kilpatrick (1957). In experiments reported by Kmetz et al. (1979), stem inoculation of soybean plants with Phomopsis species did not give rise to pod or seed infection and it was suggested that pod infections occur independently of stem infections. Also, Dhingra & Sinclair (1973) reported variability in M. phaseolina isolates obtained from a single soybean plant, raising the possibility that multiple systemic and/or aerial plant infections had occurred. Electrophoretic genetic markers such as RFLPs and RAPDs can be used to discriminate between multiple isolates of plant pathogenic and endophytic fungi obtained from single lesions and leaves. RFLP analysis of nuclear ribosomal DNA (rDNA) of Alternaria alternata isolates obtained from single black spot lesions on Japanese pear (Pyrus pyrifolia) leaves led to the conclusion that co-infection by different isolates of the fungus was responsible for the variable rDNA types (Adachi & Tsuge, 1994). Similarly, RAPD analysis was used in the identification of up to four distinct individuals of Discula umbrinella in a single beech (Fagus sylvatica) leaf and led to the conclusion that multiple infections were responsible for this observation (Haemmerli et al., 1992).

Anecdotal evidence from commercial mungbean growers suggested that high levels of Macrophomina infection in harvested seed were associated with rainfall during the early pod filling stages. Fuhlbohm (2004) isolated M. phaseolina from soil peds that had been splashed onto leaves of mungbean plants growing in the field after several rainfall events, and from seeds inside pods with adherent soil particles. In this study, inoculations of in planta and excised green pods with aqueous suspensions of pure microsclerotia inoculum of M. phaseolina resulted in significant levels of seed infection only when there were two or more days of pod wetness. These results suggest that microsclerotia of M. phaseolina transported to the surfaces of green developing pods in soil peds splashed from the ground during rain events, or in windblown soil particles (Fuhlbohm, 2004), are a significant source of inoculum leading to seed infection. Although the role of pycnidiospores in pod and seed infection was not studied by Fuhlbohm (2004), it is possible that they may play a role under field conditions. Luttrell & Garren (1952) demonstrated that pycnidiospores of M. phaseolina sprayed onto snap bean (Phaseolus vulgaris) plants caused stem and leaf lesions. Some isolates of M. phaseolina readily produce pycnidia on infected plant parts (Dhingra & Sinclair, 1978), and Fuhlbohm (2004) observed pycnidia on all aboveground plant parts, albeit rarely. The pycnidiospores of M. phaseolina exude from pycnidia in a mucilaginous cirrhus (Fitt et al., 1989), so would rely on water splash or physical transport for their dispersal, as is the case for Ascochyta rabiei (Bayaa & Chen, 2011). Consequently, during rainy weather, infection of mungbean seed may result from both microsclerotial and conidial inoculum being transported to the surfaces of green pods.

The possibility that seed infection could result from systemic growth of M. phaseolina within mungbean plants under a particular set of conditions cannot be entirely discounted, as there have been several reports which suggest that systemic infection by the pathogen occurs in other hosts. Although Gangopadhyay et al. (1970) reported that M. phaseolina was present in seed harvested from soybean plants after inoculations of the nodal, internodal and hypocotyl regions prior to flowering, the mode of seed colonization was not described. Infected sunflower seed was produced on plants growing in soil artificially infested with M. phaseolina (Fakir et al., 1976), but no infected seed was produced on plants grown from naturally infected planting seed. In both these reports details on the growing conditions of the plants are scant, so they may have been subjected to infection by M. phaseolina from sources other than the inoculum used in the inoculations. In this regard, the finding that an aerial application of Spray.Seed®250, a herbicide commonly used in Australia to desiccate crops prior to harvest, can contribute to colonization of mungbean seed by M. phaseolina is significant. When M. phaseolina inoculum was placed within 130 mm of gynophores and the plants sprayed with the herbicide, systemic colonization of the peduncle, pod and seed occurred.

The management of charcoal rot on crops such as sorghum, maize, soybean and mungbean is difficult due to the pathogen's wide host range, its ability to colonize the roots of many hosts without causing symptoms (Fuhlbohm et al., 2012) and the long survival of the microsclerotia (Dhingra & Sinclair, 1978). Strategies including planting at the optimum density, regular irrigation to minimize abiotic stress, rotations and plant resistance have been used to manage charcoal rot in some crops, with varying degrees of success. However, the management of seedborne infection by M. phaseolina has not been extensively studied. Other significant seedborne fungi that infect through the developing pods or fruits are managed by plant resistance, the application of aerial fungicide sprays (Kulik & Sinclair, 1999), or by heat treatment of harvested seed (Neergaard, 1979). Fuhlbohm (2004) investigated some of these options, using applications of the fungicide carbendazim on developing pods and heat treatment of seed at temperatures and times ranging from 45 to 70°C and 1 to 10 min, but could never completely eliminate M. phaseolina from the seed without causing a significant reduction in germination. At present, the only practical management option for minimizing seedborne Macrophomina is the avoidance of fields suspected of having high levels of inoculum. Future studies may identify mungbean germplasm that has resistance to infection of pods and seeds by M. phaseolina.

Acknowledgements

This paper is dedicated to Dr Michael John Fuhlbohm, friend and colleague who died under tragic circumstances on 28 April 2008. The other authors wish to thank the Grains Research and Development Corporation for a Grains Research postgraduate scholarship for Michael, the former Botany Department, University of Queensland and the Cooperative Research Centre for Tropical Plant Pathology for financial and resource support, and Michael's family and friends for their moral support during his PhD studies.

Ancillary