Foliar Application of Fungal Biocontrol Agents for the Control of White Mold of Dry Bean Caused by Sclerotinia sclerotiorum

Field experiments were conducted during 1992–1994 to evaluate the effectiveness of five indigenous fungi for control of white mold (Sclerotinia sclerotiorum) of dry bean (Phaseolus vulgaris). The five fungi consisted of one antagonist, Epicoccum purpurascens, and four mycoparasites, Coniothyrium minitans, Talaromyces flavus, Trichothecium roseum, and Trichoderma virens. Spore suspensions of each fungus were sprayed onto bean plants two or three times during the early bloom to midbloom period. Incidence of white mold of dry bean was significantly reduced by all biocontrol agents. C. minitans and E. purpurascens, the most effective agents, reduced the proportion of plants infected by an average of 56 and 43%, respectively (P < 0.001). C. minitans was the only biocontrol agent recovered consistently from sclerotia and diseased seed present in harvested samples. It was recovered at similar frequencies in samples from all treatments. Of the sclerotia of S. sclerotiorum collected from harvested seed, 59% were infected by C. minitans in 1993 and 20% were infected by C. minitans in 1994. In three additional trials in 1994, comparing C. minitans with the fungicide benomyl, the fungus was not effective in any of the experiments, whereas benomyl reduced disease incidence relative to the control in one trial. The study suggests that, among the five indigenous fungi, C. minitans is the most promising agent for control of white mold of dry bean under Canadian prairie conditions.     

Selection of strains of Epicoccum purpurascens for tolerance to fungicides and improved biocontrol of Sclerotinia sclerotiorum

A wild-type isolate of Epicoccum purpurascens was exposed to shortwave ultraviolet light. One of the resulting cultures (M-20-A) was grown on media amended with the fungicides iprodione or vinclozolin and fungicide-tolerant strains were obtained. Several comparisons were made between new strains and the wild type. Sporulation was improved compared with the wild type. Strains varied in their tolerance to iprodione and vinclozolin but were not tolerant to the fungicide benomyl. Strains R4000, 16-B, and 7-A inhibited Sclerotinia sclerotiorum in vitro more than either the wild type or M-20-A, and exhibited improved control of white mold of bean in the greenhouse compared with M-20-A. Key words: biological control, fungicide resistance, white mold, iprodione, vinclozolin.     

Mycoflora and Potential for Mycotoxin Production of Freshly Harvested Black Bean from the Argentinean Main Production Area

A mycological survey was carried out, for the first time, on black bean samples from the northwestern Argentinean province of Salta in the 1999 harvest season. Ten varieties of black beans were evaluated at three locations. Species of the genus Alternaria were the most prevalent component of the black bean mycoflora. Species of Fusarium, Sclerotinia, Rhizoctonia and Acremonium were also recorded. The predominant species of the genera isolated were Alternaria alternata, Sclerotinia sclerotiorum, Rhizoctonia solani, Fusarium semitectum and Acremonium strictum. An analysis of variance was applied to determine possible differences between black bean varieties. Variety FT88/519 was the most susceptible to Sclerotinia sclerotinia infection, while the variety DOR 604 was the least susceptible. As toxigenic species were recovered, Alternaria toxins, zearalenone and trichothecenes may pose a contamination risk for black bean.     

Development of a selective myclobutanil agar (MBA) medium for the isolation of Fusarium species from asparagus fields

A new selective myclobutanil agar medium for the detection of Fusarium, species is proposed. Ten media formulations based on various selective agents (pentachloronitrobenzene (PCNB), Rose Bengal, malachite green, sodium hypochlorite, captan, benomyl, chlorotalonil, myclobutanil, thiram, and cupric sulfate) were compared. First, mycelium growth and colony appearance of Alternaria alternata, Aspergillus flavus, Cladosporium cladosporioides, Epicoccum nigrum, Fusarium sp., Fuisarium solani, Fusarium moniliforme, Fusarium oxysporum f.sp. dianthi, Penicillium sp., and Trichoderma viride isolates were compared. Second, the ability of the different media to isolate and enumerate fusaria from asparagus fields was evaluated. The myclobutanil-based medium showed the highest selectivity to Fusarium spp. growth but required a slightly longer incubation time (>5 d) than peptone-pentachloronitrobenzene-based agar (PPA) (< 5 d). PPA allowed a faster fusaria growth but also permited the growth of other moulds. The other media were less selective and did not allow to isolate fusaria or to differenciate them from other growing fungi.     

Quantitative and qualitative studies of the microflora of barley malt production

Populations of aerobic heterotrophic bacteria, mycelial fungi and yeasts occurring in malting barley were estimated by a plate technique and scanning electron microscopy. There was an increase in the total number of micro-organisms during germination, although populations declined after kilning. Bacteria dominated on all samples, with progressively lower populations of yeasts and filamentous fungi. There was no obvious spatial distribution of micro-organisms on the samples although there appeared to be high populations of bacteria and fungal hyphae on the inner surface of the kernels. The dominant groups of aerobic heterotrophic bacteria were presumptively identified as Alcaligenes sp., Arthrobacter globiformis, Clavibacter iranicum, Erwinia herbicola, Lactobacillus spp. and Pseudomonas fluorescens. The principal filamentous fungi were identified as Aiternaria alternata, Aspergillus glaucus (group), Cladosporium macrocarpum, Epicoccum purpurascens, Fusarium avenaceum, Geotrichum candidum and Penicillium spp. The yeasts isolated most frequently were Candida catenulata, C. vini, Debaryomyces hansenii, Hansenula polymorpha, Kloeckera apiculata, Rhodotorula mucilaginosa, Sporobolomyces roseus and Trichosporon beigelii.     

Efficacy of microorganisms selected from compost to control soil-borne pathogens

Suppression of soil-borne plant pathogens with compost has been widely studied. Compost has been found to be suppressive against several soil-borne pathogens in various cropping systems. However, an increase of some diseases due to compost usage has also been observed, since compost is a product that varies considerably in chemical, physical and biotic composition, and, consequently, also in ability to suppress soil borne diseases. New opportunities in disease management can be obtained by the selection of antagonists from suppressive composts. The objective of the present work was to isolate microorganisms from a suppressive compost and to test them for their activity against soil-borne pathogens. A compost from green wastes, organic domestic wastes and urban sludge's that showed a good suppressive activity in previous trials was used as source of microorganisms. Serial diluted suspensions of compost samples were plated on five different media: selective for Fusarium sp., selective for Trichoderma sp., selective for oomycetes, potato dextrose agar (PDA) for isolation of fungi, lysogeny broth (LB) for isolation of bacteria. In total, 101 colonies were isolated from plates and tested under laboratory conditions on tomato seedlings growing on perlite medium in Petri plates infected with Fusarium oxysporum f.sp. radicis-lycopersici and compared to a commercial antagonist (Streptomyces griserovidis, Mycostop, Bioplanet). Among them, 28 showed a significant disease reduction and were assessed under greenhouse condition on three pathosystems: Fusarium oxysporum f.sp. basilica/basil, Phytophthora nicotianae/tomato and Rhizoctonia solani/bean. Fusarium spp. selected from compost generally showed a good disease control against Fusarium wilts, while only bacteria significantly controlled P. nicotianae on tomato under greenhouse conditions. None of the microorganisms was able to control the three soil-borne pathogens together, in particular Rhizoctonia solani. Results confirmed the good suppressive activity of the compost under study against soil-borne pathogens. The selection of antagonists from compost is a promising strategy for the development of new biological control agents against soil-borne pathogens.     

Constitutive expression of a celery mannitol dehydrogenase in tobacco enhances resistance to the mannitol-secreting fungal pathogen Alternaria alternata

Our previous observation that host plant extracts induce production and secretion of mannitol in the tobacco pathogen Alternaria alternata suggested that, like their animal counterparts, plant pathogenic fungi might produce the reactive oxygen quencher mannitol as a means of suppressing reactive oxygen-mediated plant defenses. The concurrent discovery that pathogen attack induced mannitol dehydrogenase (MTD) expression in the non-mannitol-containing host tobacco suggested that plants, unlike animals, might be able to counter this fungal suppressive mechanism by catabolizing mannitol of fungal origin. To test this hypothesis, transgenic tobacco plants constitutively expressing a celery Mtd cDNA were produced and evaluated for potential changes in resistance to both mannitol- and non-mannitol-secreting pathogens. Constitutive expression of the MTD transgene was found to confer significantly enhanced resistance to A. alternata, but not to the non-mannitol-secreting fungal pathogen Cercospora nicotianae. These results are consistent with the hypothesis that MTD plays a role in resistance to mannitol-secreting fungal plant pathogens.     

中国叶围煤污菌初探——Ⅲ.黄瓜叶围煤污菌的研究

对山东省黄瓜(Cucumis sativus L.)叶围煤污菌种群组成、分布、演替和对黄瓜的生理影响的研究结果表明;8个地市黄瓜叶面真菌有15属21种,其中属煤污菌的有6属10种:链格孢(Alternaria alternata Karst.),出芽短梗霉[Aureobasidium pullulans(deBary)Arn.],芽枝状枝孢[Cladosporium cladosporioides(Fres.)de Vries]尖孢枝孢(C.oxysporum Berk.＆Curt.)大孢枝孢(C.macarocarpum Preuss),多主枝孢[C.herbarum(Pers.)Link.],球孢枝孢(C.sphaerospermum Perz.),灰腐质霉(Humicola grisea Traaen.),紫附球菌(Epicoccum purpurascens Ehrenb.ex Wallr.)和弯孢霉[Curvularia lunala (Walk.)Boed.].链格孢和枝孢霉(Cladosporium spp.)是各地区主要种群.黄瓜叶面真菌的种类和密度与苗龄、叶面可溶性糖和氨基酸组分含量成极显著正相关.随着苗龄的增长,真菌的密度和种类增多.用两个主要种群的分生孢子悬浮液接种黄瓜叶面,形成明显的煤污,导致叶面光合作用降低,呼吸作用升高,气孔阻力增大,蒸腾作用下降.穗状德氏霉(Drechsleraspicifer R.Nelson)和核茎点霉(Phoma pulaminum Speg.)是国内的新记录.     

Isolation and Identification of Xanthotoxin (8-Methoxypsoralen) and Bergapten (5-Methoxypsoralen) from Celery Infected with Sclerotinia sclerotiorum

Methods for the production, isolation, and identification of xanthotoxin and bergapten from celery diseased by Sclerotinia sclerotiorum (Libert) de Bary were investigated. The only conditions under which this mold was capable of producing xanthotoxin and bergapten occurred when the mold was actively growing on fresh (metabolizing) celery. Neither compound was found in uninfected celery, in the mold growing on nutrient media, on nutrient media fortified with 30% filter-sterilized celery juice, or on nonmetabolizing celery. Maximal xanthotoxin production of 320 mug per g of dry rotted celery occurred at 20 C, although mycelial growth increased until 30 C. Neither xanthotoxin nor bergapten was found when the mold grew on 11 agricultural commodities other than celery.     

Detection of fungal infectous agent of wheat grains in store-pits of Markazi province, Iran

Wheat is an economic and important crop that provides approximately 20% of food calorie in the world. It is first crop in Iran and cultivated in the most areas of this country. Store-pit fungi make undesirable changes in quality and appearance of wheat grains. Even, some fungi produce different mycotoxins which are toxic to human and livestock's that use wheat grains as source of food. In this study, several samples were randomly collected from each of five store-pits located in different areas of Markazi Province including: Arak, Mahallat, Khomein, Saveh and Sarband. Grains were treated on PDA, and blotter, agar and washing test also used for isolating and detection of fungi. At least 100 grains per each sample were randomly used for each test and treatment. The fungi that determined in this study were Cochliobolus australiensis, Cladosporium herbarum, Epicoccum sp., Tilletia leavis, Aspergillus flavus, A. niger, A. fumigatus, Alternaria alternata, Alternaria sp., Penicillium italicum, P. digitatum, Fusarium sp., Rhizopus sp., Ustilago tritici, Scytalidium sp. Among these fungi the most isolates were belonged to Cladosporium, Alternaria, Rhizopus and Fusarium. Cladosporium herbarum was the most common in different sampling areas. Tilletia laevis and Ustilago tritici were just recovered in washing test. This study revealed that different fungi are associated with wheat grains in store-pits in Markazi Province. Some of them like Aspergillus flavus normally produce aflatoxin, a very toxic and carcinogenic mycotoxin that is harmful for human.     

捕食线虫丝孢菌的菌寄生性测定

研究了节丛孢Arthrobotrys、单顶孢Monacrosporium和隔指孢Dactylella三个捕食线虫丝孢菌属16个菌株,对水稻立枯丝核菌RhizoctoniasolaniAG1、大豆核盘菌Sclerotiniasclerotiorum、茄科镰刀菌Fusariumsolani和恶疫霉Phytophthoracactorum四种常见土壤植物病原真菌的菌寄生性。结果表明供试菌可以通过弹簧式菌丝圈缠绕、类附着胞结构吸附、简单的菌丝缠绕或者贴附寄主菌丝生长四种方式寄生病原菌。其中,绝大多数菌株对立枯丝核病菌有寄生作用,一些供试真菌对其它三种病原真菌有寄生现象。利用孢子液浸泡法测定了其中5种捕食线虫真菌对核盘菌菌核的寄生能力,显示有较高寄生率。     

Mycological survey of Korean cereals and production of mycotoxins by Fusarium isolates

The fungal species isolated from Korean cereals (barley, polished barley, wheat, rye, and malt) were Alternaria spp., Aspergillus spp., Chaetomium spp., Drechslera spp., Epicoccum sp., Fusarium spp., and Penicillium spp., etc. The number of Fusarium strains isolated was 36, and their ability to produce Fusarium mycotoxins on rice was tested. Nivalenol (NIV) was produced by Fusarium graminearum (7 of 9 isolates), Fusarium oxysporum (3 of 10 isolates), and Fusarium spp. (7 of 15 isolates). Of 15 isolates of Fusarium spp., 6 formed deoxynivalenol (DON). Fusarenon-X and 3-acetyl-DON were produced by most NIV- and DON-forming isolates, respectively. Zearalenone was produced by 3 isolates of F. graminearum, 1 isolate of Fusarium equiseti, and 11 isolates of Fusarium spp. T-2 toxin was not produced by any Fusarium isolates. The highest concentrations of mycotoxins produced by Fusarium isolates were 77.4 (NIV), 5.3 (DON), 138.3 (fusarenon-X), 40.6 (3-acetyl-DON), and 23.2 (zearalenone) micrograms/g.     

Tropical soil microflora of spice-based cropping systems as potential antagonists of root-knot nematodes

Suppression of plant parasitic nematodes with nematode predators, parasites or antagonists is an eco-friendly approach than the toxic chemicals. In a study, soil borne fungi from the rhizosphere of major spice crops were collected from diverse cropping systems prevailing in three southern states of India. A series of in vitro studies were conducted using 73 freshly collected fungal isolates and 76 isolates obtained from other sources. Out of this 67 isolates were not parasitic on females of root-knot nematodes whereas 115 isolates, though colonized the egg masses, did not show any signs of parasitism on nematode eggs. Fifty-nine isolates showed 50-90% inhibition in egg hatch. Pochonia chlamydospora, Verticillium lecanii, Paecilomyces lilacinus, and few isolates of Trichoderma spp. showed >25% parasitism on root-knot nematode eggs. The most promising isolates in this study were one isolate each of Aspergillus (F.45), Fusarium (F.47), and Penicillium (F.59); three each isolates of Trichoderma (F.3, F.52, and F.60) and Pochonia (F.30 and Vc.3) Verticillium (Vl); and two isolates of fungi that could not be identified (F.28 and F.62). Parasitism by Aspergillus tamarii, Aspergillus ustus, Drechslera sp., Humicola sp., and Scopulariopsis sp. on root-knot nematode eggs or females, reported in the present study, are new reports.     

Mycological survey of Korean cereals and production of mycotoxins by Fusarium isolates.

The fungal species isolated from Korean cereals (barley, polished barley, wheat, rye, and malt) were Alternaria spp., Aspergillus spp., Chaetomium spp., Drechslera spp., Epicoccum sp., Fusarium spp., and Penicillium spp., etc. The number of Fusarium strains isolated was 36, and their ability to produce Fusarium mycotoxins on rice was tested. Nivalenol (NIV) was produced by Fusarium graminearum (7 of 9 isolates), Fusarium oxysporum (3 of 10 isolates), and Fusarium spp. (7 of 15 isolates). Of 15 isolates of Fusarium spp., 6 formed deoxynivalenol (DON). Fusarenon-X and 3-acetyl-DON were produced by most NIV- and DON-forming isolates, respectively. Zearalenone was produced by 3 isolates of F. graminearum, 1 isolate of Fusarium equiseti, and 11 isolates of Fusarium spp. T-2 toxin was not produced by any Fusarium isolates. The highest concentrations of mycotoxins produced by Fusarium isolates were 77.4 (NIV), 5.3 (DON), 138.3 (fusarenon-X), 40.6 (3-acetyl-DON), and 23.2 (zearalenone) micrograms/g.     

Mycobiota associated with larval mines of Thrypticus truncatus and T. sagittatus (Diptera, Dolichopodidae) on waterhyacinth, Eichhornia crassipes, in Argentina

Thrypticus truncatus is a candidate agent for biocontrol of waterhyacinth; the larvae of this diptera mine in the petioles and feed on the phloem in the vascular bundles. The mycobiota associated with T. truncatus and T. sagittatus mines was investigated during two surveys undertaken in the spring and autumn in the Delta of the Paraná River, Argentina. Isolations were made from the mines and larval feeding points, as well as from the larvae, following dissection of the petioles, and plated onto agar. Young and upper parts of the petioles without Thrypticus mines were used as controls. Twenty eight fungal species were isolated from the mines. Pestalotiopsis guepinii, Mucor attenuatus, Phoma tropica, Achlya americana, Fusarium avenaceum, Cladosporium cladosporioides, Clonostachys rosea, Epicoccum purpurascens, Plectosporium tabacinum, Alternaria alternata, and Acremonium sp. were the most common fungi associated with mines and feeding points. Cladosporium cladosporioides, Cytospora sp., Mucor attenuatus, and Phoma tropica were associated with the larval body. The list of fungi in mines was compared with bibliographic information to determine if the species are known pathogens on waterhyacinth or other plant species. This is the first study on mycobiota associated with T. truncatus and T. sagittatus mines in waterhyacinth petioles in Argentina.     

Assessment of diversity,distribution and antibacterial activity of endophytic fungi isolated from a medicinal plant <Emphasis Type="Italic">Adenocalymma alliaceum</Emphasis> Miers

A study was conducted for isolation, identification and antibacterial potential of fungal endophytes of Adenocalymma alliaceum Miers., (Bignoniaceae), a medicinal shrub vine plant which has long history for its usages in curing various disorders. A total of 149 isolates of endophytic fungi representing 17 fungal taxa were obtained from 270 segments (90 from each stem, leaf and petiole) of this plant. Hyphomycetes (77.85%) were the most prevalent, followed by Ascomycetes (8.05%) and Coelomycetes (4.03%) respectively. A considerable amount of fungal isolates was kept under (10.07%) Mycelia-Sterilia (MS). Leaf harboured maximum colonization of endophytic fungi (72.22%) which was greater than stem (67.78%) and petiole (25.54%). The Jc similarity index was maximum (0.619) between stem vs leaf followed by leaf vs petiole (0.571) and stem vs petiole (0.428). The dominant endophytic fungi were Alternaria alternata, Aspergillus niger, Stenella agalis, Fusarium oxysporum, Curvularia lunata and Fusarium roseum. Among twelve endophytic fungi tested for antibacterial activity, crude extracts of nine endophytic fungi (75%), showed antibacterial potential against one or more clinical human pathogens. Alternaria alternata, Curvularia lunata, Penicillium sp. and Chaetomium globosum exhibited significant antibacterial activity against 4 of 5 tested pathogens, showing broad spectrum activity. This investigation explains the value of sampling from different tissues of a host plant for the greater species diversity, and additionally, the antibacterial screening of some endophytic fungi from this specific medicinal plant may represent a unique source for many of the useful antibacterial compounds.     

Genetic and physiological response to fumonisin and AAL-toxin by intact tissue of a higher plant

The differential phytotoxicity of purified AAL-toxin to lines of tomato isogenic for the Asc gene parallels resistance to Alternaria alternata f.sp. lycopersici. This relationship, as reported earlier, is consistent with the role of AAL-toxin as a host-specific toxin with the role of a primary chemical determinant of Alternaria stem canker. Current results indicate the pathogen and the AAL-toxin also can be recovered from ripe fruit with symptoms of the disease known as black mold. Fumonisins are structurally similar to the AAL-toxins but are secreted by Fusarium moniliforme which is taxonomically distinct from A. alternata. F. moniliforme, is not pathogenic on living tomato tissues but was recovered from ripe tomato fruit with symptoms of black mold. The penetration of ripe fruit and subsequent colonization by both fungi appears to be saprophytic. Fumonisins and AAL-toxins express equivalent genotype-specific activity against the isogenic Asc lines of tomato and produce equivalent necrotic symptoms in tomato leaflet bioassays. Evidence was obtained that the biosynthetic pathway for production of these toxins is present in several species of both Alternaria and Fusarium. Toxin biosynthesis was sensitive to nutritional regulation in both genera. However, pathogenicity on tomato was not altered by the medium used for inoculum production in either genera and remained restricted to A. alternata f.sp. lycopersici in the studies reported here. Differences in the amount of toxin produced were found among isolates of both genera while the magnitude of the differences was defined by the substrate on which the fungi were grown.     

Bacillus strains isolated from rhizosphere showed plant growth promoting and antagonistic activity against phytopathogens

Seven bacterial isolates screened from rhizosphere of common bean growing at Uttarakhand Himalaya showed potential plant growth promoting (PGP) and antagonistic activities. Based on 16S rRNA gene sequence the isolate BPR7 was identified as Bacillus sp. BPR7. The strain BPR7 produced IAA, siderophore, phytase, organic acid, ACC deaminase, cyanogens, lytic enzymes, oxalate oxidase, and solubilized various sources of organic and inorganic phosphates as well as potassium and zinc. Strain BPR7 strongly inhibited the growth of several phytopathogens such as Macrophomina phaseolina, Fusarium oxysporum, F. solani, Sclerotinia sclerotiorum, Rhizoctonia solani and Colletotricum sp. in vitro. Cell-free culture filtrate of strain BPR7 also caused colony growth inhibition of all test pathogens. PGP and antifungal activities of Bacillus sp. BPR7 suggest that it may be exploited as a potential bioinoculant agent for P. vulgaris.     

Mycotoxin-producing potential of fungi associated with qat (Catha edulis) leaves in yemen

Forty-four fungal species belonging to 20 genera were isolated from 30 samples of qat leaves. The most frequent genera wereAspergillus, Alternaria, Penicillium, andCladosporium followed byFusarium, Drechslera, Chœtomium, andMucor. The most prevalent species in above genera wereAspergillus niger, A. flavus, A fumigatus, Alternaria alternata, Penicillium chrysogenum, P. citrinum, Cladosporium cladosporioides, andFusarium verticillioides. From these fungi, 17 species (39%) related to 7 genera (35%) proved to be true endophytes. Eleven out of 75 isolates were mycotoxigenic.A. alternata produced alternariol and alternariol monomethyl ether whereasA. flavus produced aflatoxins B₁ and B₂. Ochratoxin A, sterigmatocystin, citrinin and T-2 toxin were produced byA. ochraceus, A. versicolor, P. citrinum andF. oxysporum, respectively. The presence of such toxigenic fungi associated with qat leaves is considered to be a threat to public health.