Ultrastructural and Cytochemical Aspects of the Biological Control of Botrytis cinerea by Candida saitoana in Apple Fruit

ABSTRACT Biocontrol activity of Candida saitoana and its interaction with Botrytis cinerea in apple wounds were investigated. When cultured together, yeast attached to Botrytis sp. hyphal walls. In wounded apple tissue, C. saitoana restricted the proliferation of B. cinerea, multiplied, and suppressed disease caused by either B. cinerea or Penicillium expansum. In inoculated apple tissue without the yeast, fungal colonization caused an extensive degradation of host walls and altered cellulose labeling patterns. Hyphae in close proximity to the antagonistic yeast exhibited severe cytological injury, such as cell wall swelling and protoplasm degeneration. Colonization of the wound site by C. saitoana did not cause degradation of host cell walls. Host cell walls in close contact with C. saitoana cells and B. cinerea hyphae were well preserved and displayed an intense and regular cellulose labeling pattern. In addition to restricting fungal colonization, C. saitoana induced the formation of structural defense responses in apple tissue. The ability of C. saitoana to prevent the necrotrophic growth of the pathogen and stimulate structural defense responses may be the basis of its biocontrol activity.     

Effects of Ca2+ and Mg2+ on Botrytis cinerea and Penicillium expansum in vitro and on the biocontrol activity of Candida oleophila

Calcium salts have been reported to play an important role in the inhibition of postharvest decay of apples and in enhancing the efficacy of postharvest biocontrol agents. Therefore, the present study was conducted in order to examine and compare the effects of calcium and magnesium salts on the germination and metabolism of the postharvest pathogens Botrytis cinerea and Penicillium expansum , and to determine the effects of these salts on the biocontrol activity of two isolates (182 and 247) of the yeast Candida oleophila. Increasing concentrations of CaCl₂ (25–175 mM) resulted in decreased spore germination and germ-tube growth of both pathogens. The greatest effect was observed in the case of B. cinerea. The inhibitory effect could be overcome by the addition of glucose to the germination medium. MgCl₂ (25–175 mM) had no effect on germination or germ-tube growth of either pathogen, indicating that the calcium cation rather than the chloride anion was responsible for the inhibition. The pectinolytic activity of crude enzyme obtained from the culture medium of both pathogens was also inhibited by 25–175 mM CaCl₂, with the greatest effect on the crude enzyme from P. expansum. Biocontrol activity of isolate 182 was enhanced by the addition of 90 or 180 MM CaCl₂, whereas there was no effect on the biocontrol activity of isolate 247. This was apparently due to the inability of isolate 247 to proliferate in apple wounds. It is postulated that enhanced biocontrol activity of isolate 182 of the yeast C. oleophila in the presence of Ca²⁺ ions is directly due to the inhibitory effects of calcium ions on pathogen spore germination and metabolism, and indirectly due to the ability of isolate 182 to maintain normal metabolism in the presence of"toxic" levels of calcium.     

Control of postharvest pathogens and colonization of the apple surface by antagonistic microorganisms in the field

ABSTRACT Selected isolates of Aureobasidium pullulans, Rhodotorula glutinis, and Bacillus subtilis reduced the size and number of lesions on wounded apples caused by the postharvest pathogens Penicillium expansum, Botrytis cinerea, and Pezicula malicorticis. Combinations of the antagonistic microorganisms were applied to apple trees in the field late in the growing season of two consecutive years. The population dynamics of the introduced microorganisms and the incidence of fruit decay were determined. Population sizes of introduced antagonists on apple surfaces increased in the field following application of treatments until harvest. After transfer of the fruit from the field into cold storage, the populations of the introduced antagonists remained higher than in the control treatments. Identification of the applied isolates of A. pullulans and R. glutinis during the experiments was achieved by isolate-specific DNA probes generated from random amplified polymorphic DNA. A combination of two strains of A. pullulans and one strain of R. glutinis suppressed rotting of apple to the same extent as the commonly used fungicide Euparen. Our data demonstrate that the application of antagonistic microorganisms in the field represents a promising alternative to fungicide treatments to control post-harvest diseases of apple.     

Enhancement of Biocontrol Activity of Cryptococcus laurentii by Silicon and the Possible Mechanisms Involved

ABSTRACT Exogenous application of silicon (Si) in the form of sodium metasilicate reduced disease development caused by Penicillium expansum and Monilinia fructicola in sweet cherry fruit at 20 degrees C. The inhibition of fruit decay was correlated closely with Si concentrations. Silicon at concentrations of 1%, in combination with the biocontrol agent Cryptococcus laurentii at 1 x 10(7) cells per ml, provided synergistic effects against both diseases. Population dynamics of C. laurentii were stimulated by Si 48 h after the yeast treatment in the wounds of sweet cherry fruit. Silicon strongly inhibited spore germination and germ tube elongation of P. expansum and M. fructicola in vitro. Based on results with scanning electron microscopy, growth of both pathogens was significantly inhibited by Si in the wounds of sweet cherry fruit. Compared with the wounded water control, Si treatment induced a significant increase in the activities of phenylalanine ammonia-lyase, polyphenoloxidase, and peroxidase in sweet cherry fruit but did not increase the levels of lignin. Application of Si activated a cytochemical reaction and caused tissue browning near the site of wounding. Based on our studies, the improvement in biocontrol efficacy of antagonistic yeast when combined with Si may be associated with the increased population density of antagonistic yeast by Si, the direct fungitoxicity property of Si to the pathogens, and the elicitation of biochemical defense responses in fruit.     

Antagonism of Trichoderma harzianum ETS 323 on Botrytis cinerea Mycelium in Culture Conditions

ABSTRACT Previous studies have shown that the extracellular proteins of Trichoderma harzianum ETS 323 grown in the presence of deactivated Botrytis cinerea in culture include a putative l-amino acid oxidase and have suggested the involvement of this enzyme in the antagonistic mechanism. Here, we hypothesized that the mycoparasitic process of Trichoderma spp. against B. cinerea involves two steps; that is, an initial hyphal coiling stage and a subsequent hyphal coiling stage, with different coiling rates. The two-step antagonism of T. harzianum ETS 323 against B. cinerea during the mycoparasitic process in culture was evaluated using a biexponential equation. In addition, an l-amino acid oxidase (Th-l-AAO) was identified from T. harzianum ETS 323. The secretion of Th-l-AAO was increased when T. harzianum ETS 323 was grown with deactivated hyphae of B. cinerea. Moreover, in vitro assays indicated that Th-l-AAO effectively inhibited B. cinerea hyphal growth, caused cytosolic vacuolization in the hyphae, and led to hyphal lysis. Th-l-AAO also showed disease control against the development of B. cinerea on postharvest apple fruit and tobacco leaves. Furthermore, an apoptosis-like response, including the generation of reactive oxygen species, was observed in B. cinerea after treatment with Th-l-AAO, suggesting that Th-l-AAO triggers programmed cell death in B. cinerea. This may be associated with the two-step antagonism of T. harzianum ETS 323 against B. cinerea.     

甜瓜果实表面生防芽孢杆菌的类群与鉴别

 从不同甜瓜果实表面可分离到在LB培养基上生长迅速、菌落形态呈明显差异的4类革兰氏染色阳性细菌。经过生理生化指标的鉴定、菌株间16S rDNA序列和部分特异性基因序列扩增以及种间遗传距离分析,将这4类芽孢杆菌分别鉴定为解淀粉芽孢杆菌(Bacillus amyloliquefaciens)和枯草芽孢杆菌(Bacillus subtilis)的3个形态变种。这些芽孢杆菌经继代培养20代后,仍能保持稳定的菌落形态和对灰葡萄孢(Botrytis cinerea)、链格孢(Alternaria alternata)、尖孢镰刀菌(Fusarium oxysporum)、黑曲霉(Aspegillus niger)和粉红单端孢(Trichothecium roseum)等8种果蔬采后病原真菌显著且广谱的拮抗作用。结果表明,甜瓜果实表面广泛分布着具有拮抗性能的枯草芽孢杆菌和其近缘种解淀粉芽孢杆菌,可通过特定培养条件下(LB培养基上37℃培养48h)单菌落的生长速度和菌落外观形态特征快速分离并鉴别它们的类群,为开发甜瓜果实采后保鲜制剂提供理论依据。     

Characterization of fludioxonil-resistant and pyrimethanil-resistant phenotypes of Penicillium expansum from apple

Penicillium expansum is the primary cause of blue mold, a major postharvest disease of apple. Fludioxonil and pyrimethanil are two newly registered postharvest fungicides for pome fruit in the United States. To evaluate the potential risk of resistance development in P. expansum to the new postharvest fungicides, one isolate of each of thiabendazole-resistant (TBZ-R) and -sensitive (TBZ-S) P. expansum was exposed to UV radiation to generate fungicide-resistant mutants. Four fludioxonil highly-resistant mutants (EC(50) > 1,000 microg/ml) and four pyrimethanil-resistant mutants (EC(50) > 10 microg/ml) were tested for sensitivities to thiabendazole, fludioxonil, and pyrimethanil, and fitness parameters including mycelial growth, sporulation on potato dextrose agar (PDA), sensitivity to osmotic stress, and pathogenicity and sporulation on apple fruit. The stability of resistance of the mutants was tested on PDA and apple fruit. Efficacy of the three fungicides to control blue mold incited by the mutants was evaluated on apple fruit. Six fungicide-resistant phenotypes were identified among the parental wild-type isolates and their mutants based upon their resistance levels. All four fludioxonil highly-resistant mutants were sensitive to pyrimethanil and retained the same phenotypes of resistance to TBZ as the parental isolates. All four pyrimethanil-resistant mutants had a low level of resistance to fludioxonil with a resistance factor >15. The two pyrimethanil-resistant mutants derived from a TBZ-S isolate became resistant to TBZ at 5 microg/ml. After 20 successive generations on PDA and four generations on apple fruit, the mutants retained the same phenotypes as the original generations. All mutants were pathogenic on apple fruit at both 0 and 20 degrees C, but fludioxonil highly-resistant mutants were less virulent and produced fewer conidia on apple fruit than pyrimethanil-resistant mutants and their parental wild-type isolates. Compared with the parental isolates, all four fludioxonil highly-resistant mutants had an increased sensitivity to osmotic stress on PDA amended with NaCl, while the pyrimethanil-resistant mutants did not. Pyrimethanil was effective against blue mold caused by fludioxonil-resistant mutants at both 0 and 20 degrees C. Pyrimethanil and fludioxonil reduced blue mold incited by pyrimethanil-resistant mutants during 12-week storage at 0 degrees C but were not effective at 20 degrees C. TBZ was not effective against pyrimethanil-resistant mutants derived from TBZ-S wild-type isolates at room temperature but provided some control at 0 degrees C. The results indicate that: (i) a fitness cost was associated with fludioxonil highly resistant mutants of P. expansum in both saprophytic and pathogenic phases of the pathogen but not pyrimethanil-resistant mutants; (ii) pyrimethanil possessed a higher risk than fludioxonil in the development of resistance in P. expansum; and (iii) triple resistance to the three apple-postharvest fungicides could emerge and become a practical problem if resistance to pyrimethanil develops in P. expansum populations.     

Alternative disease control agents induce resistance to blue mold in harvested 'red delicious' apple fruit

ABSTRACT Alternative control agents, including UV-type C (254 nm) irradiation, yeasts antagonistic to fungal growth, chitosan and harpin, were evaluated for their ability to induce resistance in cv. Red Delicious apple fruit against postharvest blue mold caused by Penicillium expansum. Freshly harvested and controlled atmosphere (CA)-stored fruit were treated with these agents at different doses and concentrations or with paired combinations of the agents. Treated fruit were inoculated with P. expansum 24, 48, or 96 h following treatment, and stored at 24 degrees C in the dark. The fruit were evaluated for development of disease every 2 days for 14 days by measuring the diameter of lesions that formed. The area under the disease progress curve (AUDPC) was calculated and analyzed statistically. All treatments were effective in reducing the AUDPC; UV-C was most effective, followed by harpin, chitosan, and the yeasts, respectively. Regardless of treatment, fresh fruit were more responsive to treatments than CA-stored fruit. There was a clear time-dependent response of the fruit to the treatments, in which treatments applied 96 h before inoculation provided the best results. In a few situations, the combinations of agents did provide an additive effect, but no synergistic effects were detected. Moreover, disease severity in fruit treated by any combination was markedly better than that in the controls. Although the combinations of treatments was overall less effective than the single treatments, they did provide significant reductions of the progress of disease in comparison with the controls. Because the fungus did not come into contact with any of the control agents, this study showed conclusively that the agents studied were able to induce resistance in the fruit rather than merely inhibit the pathogen directly. It also showed, for the first time, that harpin is able to induce resistance in harvested apple fruit. The use of these control agents may minimize the costs of control strategies and reduce the risks associated with the excessive use of fungicides in harvested apple fruit.     

The postharvest phase: Emerging technologies for the control of fungal diseases

Postharvest biological treatment entails a range of different approaches, including strengthening of the commodity’s natural defense mechanisms and application of antagonistic microorganisms and natural antimicrobial substances. Postharvest biological treatment has highlighted the potential of antagonistic microorganisms (fungi, bacteria and yeasts) against a limited number of pathogens, and only on specific hosts. Further studies are therefore required to identify antagonists with a broad spectrum of activity. The resistance of fruits and vegetables to postharvest diseases is closely linked to the ripening process, and drops markedly with the onset of tissue senescence: It is now possible to protect the product by inducing disease resistance. Plants produce a large number of secondary metabolites with antimicrobial effect on the main postharvest pathogens. Detailed studies have been conducted on aromatic compounds, essential oils and volatile substances. Combination of the above complementary techniques could well lead to effective control of postharvest fungal diseases.     

Characterizing the mechanism of biological control of postharvest diseases on fruits with a simple method to study competition for nutrients

ABSTRACT Biocontrol agents may compete with pathogens for nutrients and space to delay or prevent decay of fruits after harvest. These mechanisms of biological control have been difficult to study because no method has been available to determine the significance of each of the components of competition. We developed a nondestructive method using tissue culture plates with cylinder inserts containing defusing membrane at one end to study competition for nutrients without competition for space. Other biocontrol mechanisms in which direct contact between an antagonist and a pathogen is not required also can be studied. The method was used to determine the competition between the yeastlike biocontrol agent, Aureobasidium pullulans, and Penicillium expansum for limited nutrients in apple juice during 24 h incubation, simulating a fruit wound. The antagonist depleted amino acids and inhibited germination of P. expansum conidia. Exposing these conidia to fresh apple juice increased conidial germination to the level comparable to that exhibited by conidia which were not exposed to the antagonist. Because the culture plate method was nondestructive, follow-up experiments in an agar diffusion test were conducted. Juice in which the antagonist grew did not inhibit germination of P. expansum conidia that were seeded on the plates. This corroborates findings from the culture plate method that inhibition of the conidia germination resulted from competition for nutrients. The new method can be coupled with existing techniques to improve understanding of antagonist-pathogen interaction for biological control of postharvest diseases.     

野生药用植物内生真菌的分离及拮抗菌株筛选

为挖掘生防新资源,采用组织分离法从21种野生药用植物不同组织部位分离纯化内生真菌,以6种植物病原菌为靶标菌筛选拮抗菌株,并根据形态学及分子生物学对菌株进行鉴定,在此基础上研究了高效拮抗菌株对靶标菌菌丝生长及孢子萌发的影响。结果表明:从分离纯化的478株内生真菌中筛选出11株高活性拮抗菌株,分属于青霉属Penicillium、平脐蠕孢属Bipolaris、棘壳孢属Pyrenochaeta、镰孢属Fusarium、粒毛盘菌属Lachnum、垫壳孢属Coniella和Neonectria 7个属,其中青霉属占比最高,达36.36%。平皿对峙试验表明, 棘壳孢菌12-R-5对灰葡萄孢表现出高效性和专一性,其对菌丝抑制率达66.67%。含药平皿试验表明,菌株12-R-5发酵滤液10倍稀释液对灰葡萄孢菌丝生长抑制率达100%,与其他处理差异显著。菌株12-R-5 10倍发酵滤液处理灰葡萄孢10 h,孢子不萌发;100倍稀释液处理对孢子萌发抑制率为91.01%。显微镜观察发现,棘壳孢菌使灰葡萄孢菌丝扭曲变形,膨大肿胀及断裂,部分菌丝发生抑缩、消融现象。说明棘壳孢菌12-R-5菌株具有很好的生防潜力及应用前景。     

Biocontrol Potential of Metchnikowia pulcherrima Strains Against Blue Mold of Apple

ABSTRACT Eight strains of Metschnikowia pulcherrima isolated over a 4-year period from an unmanaged orchard and selected for their biocontrol activity against blue mold (caused by Penicillium expansum) of apples were characterized phenotypically, genetically, and for their biocontrol potential against blue mold on apples. All strains grew well and only differed slightly in their growth in nutrient yeast dextrose broth medium at 1 degrees C after 216 h, but large differences occurred at 0 degrees C, with strain T5-A2 outgrowing other strains by more than 25% transmittance after 360 h. This strain was also one of the most resistant to diphenylamine (DPA), a postharvest antioxidant treatment. All strains required biotin for growth in minimum salt (MS) medium, although strain ST2-A10 grew slightly in MS medium containing riboflavin or folic acid, as did ST3-E1 in MS medium without vitamins. None of the strains produced killer toxins against an indicator strain of Saccharomyces cerevisiae. Analysis of Biolog data from YT plates for all eight strains using the MLCLUST program resulted in separation of the strains into one major cluster containing four strains and four scattered strains from which strain ST1-D10 was the most distant from all other strains. This was particularly apparent in 3-D and principle component analysis. Genetic differentiation of the eight strains using maximum parsimony analysis of nucleotide sequences from domain D1/D2 of nuclear large subunit (26S) ribosomal DNA resulted in detection of two clades. Strain ST1-D10 grouped with the type strain of M. pulcherrima but the remaining seven strains grouped separately, which might possibly represent a new species. All strains significantly reduced blue mold on mature Golden Delicious apples during 1 month of storage at 1 degrees C followed by 7 days at room temperature, but strains T5-A2 and T4-A2 were distinctly more effective under these conditions. Strain T5-A2 also was the most effective in tests on harvest mature apples treated with the lowest concentration of the antagonist and stored for 3 months at 0.5 degrees C. Populations of all eight strains increased in apple wounds by approximately 2 log units after 1 month at 1 degrees C followed by 5 days at 24 degrees C. Our results indicate that M. pulcherrima is an excellent candidate for biological control of postharvest diseases of pome fruit. The variation in phenotypic, genetic, and biocontrol characteristics among strains of M. pulcherrima isolated from the same orchard should make it possible to select antagonists with characteristics that are most desirable for postharvest application.     

Antifungal Activity of 2-Deoxy-D-Glucose on Botrytis cinerea, Penicillium expansum, and Rhizopus stolonifer: Ultrastructural and Cytochemical Aspects

ABSTRACT The effect of 2-deoxy-D-glucose on major postharvest pathogens was investigated at the ultrastructural and cytochemical level. Hyphae of Botrytis cinerea, Penicillium expansum,, and Rhizopus stolonifer grown in the absence of 2-deoxy-D-glucose were normal and showed no apparent cytological alterations. In the presence of 2-deoxy-D-glucose, however, these fungi exhibited severe cellular injuries ranging from cell wall disruption to cytoplasm disintegration. Although 2-deoxy-D-glucose caused cytoplasmic degeneration in the three fungi tested, cell wall alterations were exhibited only by B. cinerea and R. stolonifer. In the latter, the retraction of degenerated cytoplasm was often accompanied by the deposition of amorphous material in paramural spaces. Cytochemical study of fungal cell wall components showed that 2-deoxy-D-glucose caused a marked increase of chitin- and beta-1,3-glucan-labeling in R. stolonifer and B. cinerea, indicating an interference of 2-deoxy-D-glucose with fungal wall biosynthesis. The observed cellular alterations indicate that 2-deoxy-D-glucose may also have affected other metabolic processes.     

Improved Control of Postharvest Decay of Pears by the Combination of Candida sake (CPA-1) and Ammonium Molybdate

ABSTRACT The potential enhancement of Candida sake (CPA-1) by ammonium molybdate to control blue and gray mold caused by Penicillium expansum and Botrytis cinerea, respectively, on Blanquilla pears was investigated. In laboratory trials, improved control of blue and gray molds was obtained with the application of ammonium molybdate (1, 5, 10, and 15 mM) alone or in combination with C. sake at 2 x 10(6) or 2 x 10(7) CFU ml(-1) on Blanquilla pears stored at 20 degrees C. In semicommercial trials at 1 degrees C for 5 months, the efficacy of C. sake at 2 x 10(6) CFU ml(-1) on reducing P. expansum and B. cinerea decay was enhanced more than 88% with the addition of ammonium molybdate 5 mM in the 1999-2000 season. In two seasons, the performance C. sake at 2 x 10(6) CFU ml(-1) plus ammonium molybdate was similar to or greater than that of C. sake at 2 x 10(7) CFU ml(-1). Similar control of blue mold was obtained on pears stored under low oxygen conditions. The preharvest application of ammonium molybdate did not reduce postharvest blue mold decay. The population of C. sake on pear wounds significantly decreased in the presence of ammonium molybdate 1 and 5 mM at 20 and 1 degrees C.     

Effect of the Biocontrol Yeast Rhodotorula glutinis Strain LS11 on Patulin Accumulation in Stored Apples

ABSTRACT Contamination of apples (Malus domestica) and derived juices with fungicide residues and patulin produced by Penicillium expansum are major issues of food safety. Biocontrol agents represent an alternative or supplement to chemicals for disease control. Our data show that these microbes could also contribute to actively decreasing patulin accumulation in apples. Three biocontrol agents, Rhodotorula glutinis LS11, Cryptococcus laurentii LS28, and Aureobasidium pullulans LS30, were examined for their in vitro growth in the presence of patulin and for their capability to decrease mycotoxin recovery from the medium. Strain LS11 yielded the highest growth rates and the greatest decrease of toxin recoveries. Further, it caused the appearance of two major spots on thin-layer chromatography (TLC) plates, suggesting possible metabolization of the mycotoxin. In vivo, i.e., in the low percentage of LS11-pretreated apples infected by P. expansum, patulin accumulation was significantly lower than in nontreated infected fruits. Yeast cells survived and increased in infected apples and, in a model system emulating decaying apple, resulted in accelerated breakdown of patulin and the production of the same TLC spots as those detected in vitro. These data suggest that biocontrol yeast cells surviving in decaying apples could metabolize patulin and/or negatively affect its accumulation or synthesis. To our knowledge, this is the first report describing the effect of a biocontrol agent on patulin accumulation in vivo.     

Control of Postharvest Decay of Apple Fruit with Candida saitoana and Induction of Defense Responses

ABSTRACT The ability of Candida saitoana to induce systemic resistance in apple fruit against Botrytis cinerea was investigated. To separate the antagonistic activity of C. saitoana from its ability to induce resistance, the antagonist and the pathogen were applied in spatially separated wounds. In fresh apples, C. saitoana applied 0 or 24 h before inoculation with B. cinerea showed no effect on lesion development caused by B. cinerea. When applied 48 to 72 h preinoculation with B. cinerea, however, C. saitoana reduced lesion diameter by more than 50 and 70%, respectively, compared with wounding. C. saitoana had no effect on lesion development on stored apples, regardless of the lag period between yeast treatment and inoculation with B. cinerea. In addition to inducing systemic resistance, C. saitoana increased chitinase and beta-1,3-glucanase activities with a higher accumulation in fresh than in stored apples. In fresh apples, the onset of systemic resistance to B. cinerea coincided with the increase in chitinase and beta-1,3-glucanase activity in systemically protected tissue. These studies show that C. saitoana is capable of inducing systemic resistance in apple fruit and indirectly suggest that antifungal hydrolases are involved in the observed systemic protection.     

抑制黄瓜枯萎病的链霉菌S-101及其生防作用研究

为了获得抑制黄瓜枯萎病的高效生防菌,对青海湖附近植被根系土壤中的微生物进行分离筛选。测定拮抗菌的抑菌活性及其对黄瓜枯萎病的防控效果。本文通过平板对峙法分离筛选出一株生防链霉菌S-101,该菌株对供试的几种病原真菌都有较好的拮抗作用,其中对尖镰孢菌黄瓜专化型的抑制率为53.3%,对草莓炭疽菌的抑菌率为45.5%;并通过形态特征及16S rDNA序列分析对菌株S-101初步鉴定为Streptomyces luridus;盆栽试验结果表明,菌株S-101发酵液对黄瓜枯萎病的防治效果达57.11%;通过构建绿色荧光蛋白标记的基因工程菌S-101-GFP,检测了该菌株在黄瓜根部及根围土壤中的定殖能力,随着定殖天数的增加,数量由处理时的1×10⁸ cfu/g逐渐减少到1×10⁶ cfu/g,0～14 d下降较快,21～28 d下降较慢,28～35 d趋于稳定,孢子数维持在1×10⁶ cfu/g。因此,菌株S-101是防治黄瓜枯萎病的潜在生防菌株,具有开发成为微生物农药的应用价值。     

Bioassays of glucosinolate-derived isothiocyanates against postharvest pear pathogens

The present study assayed the effect of six isothiocyanates (ITCs), produced by the enzymatic hydrolysis of glucosinolates, on fungal pathogens of pear. Sample pear fruits were artificially inoculated through induced wounds with conidial suspensions of Botrytis cinerea Rhizopus stolonifer Monilinia laxa Mucor piriformis or Penicillium expansum and were then treated with ITCs. Of the six ITCs tested, the ITC from glucoraphenin showed the highest effectiveness after 6 days at 20°C, against M. laxa B. cinerea and M. piriformis . The effectiveness of the ITC from glucoraphenin against M. laxa was assayed in two further trials to test the effect of ITC concentration on different concentrations of inoculum and to determine the duration of the curative effect of this ITC. ITC concentration directly affected fungus control capacity. The highest ITC concentration (3.6 mg mL⁻¹) afforded pathogen control at the highest level of pathogen concentration (10⁶ conidia mL⁻¹) after 6 days at 20°C. Its curative effect was evident up to 40 h after inoculation.     

灰葡萄孢拮抗细菌的筛选

采集扬州、淮阴两地１６种常见植物根部土壤、根和叶片样本１８２份,分离纯化后得到细菌６３２株,抑菌圈法测定对灰葡萄孢有拮抗性的５８株,拮抗性稳定的１３株。其中拮抗性最强的Ｙ２－１１－１菌株为芽孢杆菌,抑菌圈直径达２９．７ｍｍ。接种试验表明,Ｙ２－１１－１以番茄叶片和果实灰霉病的防效分别达６８．７％和７５．０％。部分菌株对其他重要植物病原真菌也有一定的拮抗作用。     

Characterization of the Exoglucanase-Encoding Gene PaEXG2 and Study of Its Role in the Biocontrol Activity of Pichia anomala Strain K

ABSTRACT The PaEXG2 gene, encoding an exo-beta-1,3-glucanase, was isolated from the biocontrol agent Pichia anomala strain K. PaEXG2 has the capacity for coding an acidic protein of 427 amino acids with a predicted molecular weight of 45.7 kDa, a calculated pI of 4.7, and one potential N-glycosylation site. PaEXG2 was disrupted by the insertion of the URA3 marker gene, encoding orotidine monophosphate decarboxylase in strain KU1, a uracil auxotroph derived from strain K. Strain KU1 showed inferior biocontrol activity and colonization of wounds on apples, compared to the prototrophic strain. Antagonism and colonization were recovered after the restoration of prototrophy by transformation with the URA3 gene. Integrative transformation was shown to be mostly ectopic in strain K descendants (only 4% of integration by homologous recombination). PaEXG2 disruption abolished all detectable extracellular exo-beta-1,3-glucanase activity in vitro and in situ but did not affect biocontrol of Botrytis cinerea on wounded apples.