An enzyme immunoassay of phaseolinone and its application in estimation of the amount of toxin in Macrophomina phaseolina-infected seeds.

A microtiter plate-based enzyme immunoassay has been developed for phaseolinone, a phytotoxin isolated from the culture filtrate of the plant-pathogenic fungus Macrophomina phaseolina (Tassi) Goid. The smallest amount of phaseolinone detectable by the method is 5 pg per well. The method is validated by comparison with high-performance liquid chromatography and used to confirm and estimate phaseolinone production in seeds infected with the fungus. The degree of seed inhibition correlated well with the amount of toxin produced in infected seeds, 50% inhibition being observed at a toxin concentration of 0.60 micrograms/g of wet tissue.     

Inhibition of seed germination by Macrophomina phaseolina is related to phaseolinone production

The production of phaseolinone, a phytotoxic metabolite of Macrophomina phaseolina in infected Phaseolus mungo seeds grown on soil, was estimated by enzyme-linked immunosorbent assay and HPLC. The degree of inhibition of seed germination correlated well with the amount of toxin produced; 50% inhibition was observed at a toxin level of 2.1 μg g^-1 of wet tissue. A comparison of the toxin-producing ability of nine isolates of the fungus obtained from different hosts and localities showed that the strain MPK'83 produced a significantly larger amount of the toxin, both in liquid culture and in infected seeds. The virulence of the isolates was related to their ability to produce phaseolinone.     

Avirulent mutants ofMacrophomina phaseolina andAspergillus fumigatus initiate infection inPhaseolus mungo in the presence of phaseolinone; levamisole gives protection

To evaluate the role of phaseolinone, a phytotoxin produced by Macrophomina phaseolina, in disease initiation, three nontoxigenic avirulent mutants of the fungus were generated by UV-mutagenesis. Two of them were able to initiate infection in germinating Phaseolus mungo seeds only in the presence of phaseolinone. The minimum dose of phaseoli-none required for infection in 30% seedlings was 2 5 mg/ml. A human pathogen, Aspergillus fumigatus was also able to infect germinating seeds of P. mungo in the presence of 5 mg/ml concentration of phaseolinone. Phaseolinone seemed to facilitate infection by A. fumigatus, which is not normally phytopathogenic, by reducing the immunity of germinating seedlings in a nonspecific way. Levamisole, a non-specific immunopotentiator gave protection against infection induced by A. fumigatus at an optimum dose of 50 mg/ml. Sodium malonate prevented the effects of levamisole.     

Differential Responses of Guayule (Parthenium argentatum Gray) Genotypes to Culture Filtrate and Toxin from Macrophomina phaseolina (Tassi) Goidanich

The potential for using cell-free culture filtrate (CFCF) and toxin (phaseolinone) from Macrophomina phaseolina for rapid and effective screening procedures for charcoalrot resistance in guayule (Parthenium argentatum) germplasm was assessed. The CFCF and partially purified phaseolinone were incorporated into modified Murashige and Skoog solid medium aat the rates of 0-100% (v/v) and 0-1000 μg ml^-1 respectively. The medium pH was adjusted to 5.8 before solidifying with 0.8% agar. Fourweek-old seedlings of 10 guayule genotypes were plantanted in the medium, incubated and rated for phytotoxic symptoms and tissue damage over a 15-day period. In a green-house study, seedling growth, phytotoxicity and damage severity were compared in 12-week-old guayule seedlings root-inoculated with M. phaseolina microsclerotia. There were significant differences (P = 0.05) in genotypic responses to the fungus, the filtrate and the toxin inoculations. Time until phytotoxic symptoms developed was inversely related to the concentrations of CFCF and the toxin. Phytotoxic symptoms were produced 6 days after exposure to 50% CFCF and 48 h after exposure to 1000μg ml^-1 of partially purified phaseolinone. A comparison of photomicrographs of the control and toxintreated root tissues revealed no damage to the control roots and extensive damage to epidermal layers of the treated roots, which was evident 48 h after exposure to 100μg ml^-1 level of phaseolinone. Significant correlations were found between tolerance to the fungus and insensitivity to the culture filtrate (r = 0.89, P = 0.05) and the toxin (r = 0.95, P = 0.001) suggesting the possibility of screening for resistance to M. phaseolina using CFCF or phaseolinone. The genotypic reactions to the CFCF were also correlated with reactions to the toxin (r = 0.90, P = 0.05). Guayule breeding lines‘UC101 and‘P3-1 exhibited the greatest tolerance to the pathogen and insensitivity to the CFCF or the toxin whereas‘Ca16′,‘Cal7′,‘N576′,‘N9-5′, 11605’and‘N6-5’were very susceptible to the pathogen and sensitive to the CFCF or the toxin.     

A new antileishmanial compound, phaseolinone

Inclusion of phaseolinone, a newly described mycotoxin, at 20 micrograms per ml in a solid culture medium (blood agar overlay) and at 50 micrograms per ml in a liquid culture (medium 199) inhibited the growth of L. donovani promastigotes. About 90% of the motile promastigotes lost motility after exposure to 50 micrograms per ml of phaseolinone for 6-7 h and here 3-day-old culture was more sensitive than 7-day-old culture. In an in vitro assay, DNA dependent RNA polymerase activity of 3-day-old promastigotes was considerably inhibited in the presence of this toxin. Therefore, this key enzyme was suggested to be one of the sites of action of phaseolinone.     

In vivo events associated with entomopathology of Beauveria bassiana for the corn earworm (Heliothis zea)

When conidia of Beauveria bassiana are injected into the hemocoel of corn earworm larvae, it appears that a positive correlation exists between exocellular proteolytic activity of the fungus and entomopathological manifestations. Once inside the hemolymph, defense mechanisms (including phagocytosis) are incapable of overcoming the fungus and one important event in a terminal mycocidal cascade involves preferential invasion of the gut wall. Such invasion helps explain the observed inhibition of feeding by infected larvae. Although histopathological changes seen in gut tissues suggest that a gut toxin is produced, evidence for such a toxin could not be obtained in preliminary tests. Biochemical changes are seen in hemolymph components; however, these are viewed as being due to general starvation rather than to specific activities of the fungus, at least up to the time that a general mycosis is established. With the host larva under physiological stress (starvation, nutrient depletion, and, possibly, toxin production in gut tissues) and failure of defense mechanisms, the infection spreads quickly and a terminal mycosis results.     

The Toxins of Helminthosporium maydis (Race T) : A Colorimetric Determination of the Toxins, Their Appearance in Culture and in Infected Plants

Host-specific toxins produced by Helminthosporium maydis, race T, are measured quantitatively by a chemical assay procedure involving reaction of the toxins with a sulfuric acidacetic anhydride reagent and measurement of the absorbance of the product at 330 nm. The assay was shown to measure total toxin concentrations after only limited fractionation of the culture medium. Using the assay it was possible to show that the highest amount of toxin per gram of fungus mycelium occurs early in the growth cycle of H. maydis. Toxins I, II, and V are the predominant toxins at these early times both in culture and in infected corn and wheat varieties. Some chromatographic and spectral properties of toxin V, a previously unreported toxin, are described. Since toxin V appears in culture prior to toxins I, II, III and IV, a precursor-product relationship can be suggested.     

Trichothecene synergism, additivity, and antagonism: the significance of the maximally quiescent ratio.

The interactive effect of the combinations of trichothecene mycotoxins often found in fungus infected plants, contaminated grain, and other biological systems is poorly understood. Growth inhibition of the yeast Kluyveromyces marxianus was used to measure the effects of HT-2 toxin, roridin A, and T-2 toxin as individual toxins or as binary mixtures. A value, the combination index, was derived which indicates the interactive effects of a binary mixture of toxins. The interaction is affected by the ratio of the individual toxins, and the percent inhibition of yeast growth. Generally the interaction of T-2 toxin and roridin A or T-2 toxin and HT-2 toxin changes from antagonistic when they cause a low percent inhibition of yeast growth to synergistic when they cause a high percent inhibition of yeast growth. Additionally, any two trichothecenes have a unique ratio, which we name the maximally quiescent ratio (or MQR), where there is the least change in the type and intensity of their interaction. The maximally quiescent ratio in this case has helped to define the nature of toxin interactions and could be used to provide insights into hormone, immune system, developmental, enzyme, and gene regulation, combined drug therapy, and the action of mixtures of natural or synthetic toxins, carcinogens, pesticides, and environmental pollutants.     

Neck rot (Botrytis allii) of bulb onions

Experiments on neck rot of onions, caused by Botrytis allii showed that, although the disease only became evident in store, a major source of the pathogen was samples of infected seeds. In 1972 and 1973, 39·5 and 71·4% respectively of commercial onion seed samples tested at Wellesbourne were infected. The pathogen was internal in seed and persisted for 3 ½ yr in infected seeds kept in a seed store at 10°C and 50% r.h. Seedlings raised from diseased seeds became infected by mycelial invasion of the cotyledon leaf tips from seed-coats many of which remained attached to the cotyledons when seedlings emerged from the soil. The fungus attacked the living tissues of these leaves symptomlessly, producing conidiophores only after the leaf tissue senesced and became necrotic. Because the fungus was symptomless, the rate of spread of the pathogen in onion crops was assessed by incubating successive samples of plants from the field in humid conditions when infected tissues developed conidiophores of the fungus. This method showed that the disease was progressive in onion crops spreading more rapidly in wet humid conditions (e.g. 1972) than in dry ones (e.g. 1973). The principal means of spread were probably fungal spores; conidiophores bearing spores being produced abundantly on plants in the field under high humidity. The fungus invaded the leaves of plants successively, first infecting each leaf at the tip and then growing downwards in the tissues and eventually invading the neck of the onion bulb via the leaves which emerged directly from the top of the neck. By harvest, the fungus was situated deep within the neck tissues of infected maturing onion bulbs.     

The field behaviour of seed-borne Ascochyta fabae and disease control in field beans

In field sowings at Cambridge 2–15% of field bean seeds carrying Ascochyta fabae produced seedlings with leaf lesions. The fungus spread for distances up to 10 m in an average season and usually infected the new crop of seed. The amount of such infection arising from a single lot varied widely when samples were grown at different centres, presumably because of differences in local weather conditions. Seed lots with approximately 1% infected seeds seem suitable for ware crop production but little or no A. fabae can be tolerated in seed intended for multiplication. Infection in British-grown commercial seed has been greatly reduced by the selection of clean seed. Health standards adopted in the Field Bean Seed Scheme may have eliminated A. fabae from one cultivar.     

The influence of colouring and pungent agents of red Chilli (Capsicum annum) on growth and aflatoxin production by Aspergillus flavus

Capsanthin and capsaicin, the colouring and pungent principles of red chilli Capsicum annum , respectively, were tested against the growth and aflatoxin producing potentials of Aspergillus flavus in SMKY liquid medium. Capsanthin completely checked both the growth and toxin production at all the concentrations viz. 0.2, 0.6 and 1.0 mg ml^-1, till the fourth day of incubation. On the 10th day growth of the fungus and toxin biosynthesis were 39 and 22% of the control, respectively, at 1.0 mg ml^-1. Capsaicin showed some inhibitory efficacy only up to the fourth day of incubation. The fungus grew thereafter with a marginal inhibition in growth at the highest concentration. The amount of the toxin in the medium was also higher.     

Dynamics of Neotyphodium endophyte infection in ageing seed pools: incidence of differential viability loss of endophyte,infected seed and non-infected seed

Symbiotic associations between grasses and vertically transmitted endophytic fungi are widespread in nature. Within grass populations, changes in the frequency of infected plants are driven by influence of the endophyte on the fitness of their hosts and by the efficiency of endophyte transmission from parent plants to their offspring. During the seed stage, the endophyte might influence the fitness of its host by affecting the rate of seed viability loss, whereas the efficiency of endophyte transmission is affected by losses of viability of the fungus within viable seeds. We assessed the viability losses of Lolium multiflorum seeds with high and low level of infection of the endophyte Neotyphodium occultans, as well as the loss of viability of the fungus itself, under accelerated seed ageing and under field conditions. Starting with high endophyte-infected accessions of L. multiflorum, we produced their low endophyte-infected counterparts by treating seeds with a fungicide, and subsequently multiplying seeds in adjacent plots allowing pollen exchange. In our accelerated ageing experiments, which included three accessions, high endophyte-infected seeds lost viability significantly faster than their low endophyte-infected counterpart, for only one accession. High endophyte-infected seeds of this particular accession absorbed more water than low endophyte-infected seeds. In contrast, the endophyte lost viability within live seeds of all three accessions, as the proportions of viable seeds producing infected seedlings decreased over time. In our field experiment, which included only one accession, high endophyte-infected seed lost viability significantly but only slightly faster than low endophyte-infected seed. In contrast, the loss of viability of the endophyte was substantial as the proportions of viable seeds producing infected seedlings decreased greatly over time. Moving the seeds from the air to the soil surface (simulating seed dispersion off the spikes) decreased substantially the rate of seed viability loss, but increased the rate of endophyte viability loss. Our experiments suggest that, in ageing seed pools, endophyte viability loss and differential seed mortality determine decreases in the proportions of endophyte-infected seeds in L. multiflorum. Endophyte viability loss within live seeds contributes substantially more to infection frequency changes than differential viability losses of infected and non-infected seeds.     

The effect of iprodione on the seed-borne phase of Alternaria brassicicola

Alternaria brassicicola infection of Brassica oleracea seeds was effectively controlled by a dust application of iprodione (Rovral 50% w.P.). At 2.5 g a.i./kg the seed-borne fungus was usually eliminated from samples with up to 61.5% affected seeds (35.5% internally diseased) but higher levels of infection required increased doses for complete eradication of the fungus. The germination of healthy seeds, including samples from 7–yr-old stocks, on filter paper was unaffected by the treatment. However, the germination of diseased samples, particularly those internally infected with A. brassicicola, was improved. More seedlings emerged from iprodione treated than from untreated seeds in glasshouse soil but the differences were not significant. The application of gamma-hexachlorocyclohexane to iprodione treated seeds sown in soil did not adversely affect subsequent emergence or disease control. Disease control was maintained and germination was not affected by the treatment when treated infected seeds were stored for 2 yr at 10 °C, 50% r.h. In a field trial iprodione seed treatment reduced seedling infection in a cabbage crop grown from naturally diseased seeds (100% contaminated, 45.5% internally infected) from 5.6 to 0.04%.     

Studies on the seed-borne phases of dark leaf spot Alternaria brassicicola and grey leaf spot Alternaria brassicaeof brassicas

Tests in Britain on samples of basic and commercial Brassica oleracea seed between 1976 and 1978 showed that many lots were infected with Alternaria brassicicola. A. brassicae was uncommon in basic seed in these years and in commercial seed harvested in 1976 and 1977 but was frequent in seed harvested in 1978. Most affected seeds were contaminated by surface-borne spores and mycelium of A. brassicicola but many were internally infected by the fungus situated within the seed-coat and in some seeds in the embryo tissues. Superficial contamination by the fungus declined rapidly after 2 yr in cabbage seeds stored at 10 °C, 50% r.h. but internal infection persisted for up to 12 yr. In some samples, internal infection was commonly associated with small shrivelled seeds. Surface contaminated and internally infected seeds transmitted the disease but seedling infection was more closely correlated with the latter.     

供给天麻种子萌发营养的真菌——紫萁小菇

从天麻种子发芽的原球茎中,共分离到12种可供给天麻种子萌发营养的真菌,其中最优良的01号菌株,诱导培养出子实体,经鉴定为紫萁小菇(Mycena osmundicola Lange),属国内新纪录。用01号菌株及紫萁小菇培养的染菌树叶,伴播天麻种子都可发芽,经酯酶同工酶凝胶电泳分析,二者酶带条数、迁移率(RF值)基本相同,证明鉴定结果可靠(标本保存在医科院药植所真菌研究室)。     

Systemic spread of downy mildew in basil plants and detection of the pathogen in seed and plant samples

Downy mildew on sweet basil (Ocimum basilicum L.) occurs worldwide. Contaminated seeds are considered as the primary inoculum source. So far no strategy to control the disease is available. Hence, the use of pathogen-free seeds is the only alternative to prevent disease outbreaks. Therefore, a rapid diagnostic method for seed testing is urgently needed. The sensitivity of a specific PCR method for direct detection of the downy mildew pathogen Peronospora belbahrii on basil samples, particularly on seeds, was evaluated. The applied PCR method proved to be very sensitive for direct detection of the pathogen on seeds and plant samples. The PCR detection limit of P. belbahrii in artificially infested seeds corresponded to the DNA amount of a single spore per seed. Additionally, the systemic spread of the pathogen from naturally infected seeds was investigated. The experiments showed that outgrowing basil plants were latently infected with the downy mildew pathogen, and the infection continued within the plant. Contaminated seeds were harvested from symptomless latently infected plants. These results support the implementation of PCR-based detection in a seed certification scheme and the necessity to control the pathogen on seeds. The PCR method can also be used for evaluation of pathogen control on seeds based on detection of the pathogen in outgrowing plants.     

Fusarium eumartii Growth in Resistant and Susceptible Oak Species

Fusarium eumartii is a fungus associated with declining Quercus robur , in which it is found in the vessels. The response of oak species to infection is known to vary: Q. robur is susceptible , but Quercus cerris and Quercus pubescens are resistant. An experiment was carried out in 1996 and repeated in 1997, to examine how F. eumartii colonization differed in oak species that were susceptible or resistant to the fungus by counting the number of vessels with mycelium at various distances from the inoculation site in infected seedlings and by determining the amount of viable fungus in infected tissue. Infected vessels with mycelium were counted on sections (10  μ m thick) cut at 0, 2, 4, 6, 8 and 10 cm from the inoculation site on 1-year-old inoculated seedlings as well as on sections cut every 2 cm to the seedling tip. The amount of viable fungus was determined by counting the colony forming units (CFUs) in stem segments from the same seedlings. Quercus robur seedlings had the greatest number of infected vessels and the greatest number of CFUs. Forty days after inoculation, the extent of vertical fungal spread was 28.12 cm in Q. robur , 3.15 cm in Q. cerris and 3.00 cm in Q. pubescens . The greatest number of CFUs was found in Q. robur at day 5 after inoculation. Analysis of variance confirmed the results.     

Selective Inhibition of K+, Na+, Cl?, and PO43? Uptake in Zea mays L. by Bipolaris (Helminthosporium) maydis Race T Pathotoxin: Evidence for a Plasmalemma Target Site? 1

Pathotoxin preparations were obtained from either axenic culture filtrate of race T of Bipolaris maydis (Nisikado) Shoemaker (new culture media and toxin purification procedures are described) or extracts of maize leaves infected with the fungus. The toxins (10(-6) to 10(-8)m) caused inhibition of [(86)Rb]K(+) uptake in leaf discs and apical root segments of Zea mays L. cv W64A Texas (Tcms) and normal (N) cytoplasms. Significant inhibition was measurable as early as 5 min after adding toxin. In Tcms per cent inhibition was increased by increasing toxin concentration and time in toxin, by using solution at pH 5 rather than pH 7, by decreasing external KCl concentration over the range 50 to 0.1 mm (in the presence of 0.5 mm CaSO(4)), or by exposing leaf discs to light rather than dark during the uptake period in toxin. Root uptake of (22)Na(+) and (36)Cl(-) was inhibited to a lesser extent than K(+). Inhibition of (32)PO(4) (3-) uptake occurred after 40 min when cyclosis had ceased.When combined with data in the literature, our data indicate that the plasmalemma is the probable primary site of toxin action in N and Tcms maize. Comparison of the effects of toxin on K(+) uptake in N and Tcms maize suggests the existence of more than one mode of toxin action: a weak disruptive effect in N and Tcms, and in addition, specific membrane sites in Tcms involved in monovalent ion uptake.Six genotypes in N or Tcms cytoplasm which exhibited different degrees of disease susceptibility in the field showed a corresponding gradation of susceptibility to the toxin when a K(+) uptake bioassay was used. This correlation is strong evidence that the sites of toxin action affecting K(+) transport have characteristics closely related to cellular factors regulating susceptibility to fungal attack.     

Pea seed infection by Mycosphaerella pinodes and Ascochyta pisi and its control by seed soaks in thiram and captan suspensions

Apparently normal pea seeds from pods bearing lesions of Mycosphaerella pinodes were often internally infected with the fungus. When infected seeds were sown in sterile grit almost all the seedlings showed disease lesions, at or below soil level, 4–6 weeks after sowing. Seed infected with Ascochyta pisi gave only 40% infection of seedlings: these showed lesions on the stem and first two leaves within 4 weeks of sowing. Infection of seeds by both pathogens could be eradicated by soaking the seeds for 24 hr. in 0.2% suspensions of thiram or captan at 30d?C. In laboratory or greenhouse tests these treatments did not check germination, but in the field the captan treatment reduced emergence. The treated seeds became fully imbibed but could be dried and stored: the thiram treatment was used for semi-commercial treatment of quantities of seed up to 3 cwt.     

Infection of Spathoglottis plicata (Orchidaceae) seeds by mycorrhizal fungus

Spathoglottis plicata seeds were encapsulated in 4-mm-diameter capsules of alginate-chitosan or alginate-gelatin and infected with the mycorrhizal fungus Rhizoctonia AM9. The encapsulated seeds were placed directly on Rhizoctonia culture. About 66% of the seeds encapsulated in sucrose-free chitosan-alginate established a symbiotic relationship with the mycorrhizal fungus after co-culturing for 2 weeks. The highest percentage of infection observed was about 84%. Addition of sucrose or using gelatin-alginate for encapsulation reduced the percentage of infection by about half. The growth of Rhizoctonia AM9 in sucrose-free alginate, chitosan and gelatin was found to be minimal. The advantages of germinating orchid seeds, encapsulated in sucrose-free polymers, through mycorrhizal infection is discussed. Received: 19 February 1998 / Revision received: 8 May 1998 / Accepted: 20 May 1998