绿木霉ZBS6黏粒基因组文库的构建及质量鉴定

为开展绿木霉ZBS6(Trichoderma viride ZBS6)拮抗基因筛选及克隆工作,建立了高质量基因组DNA的提取方法,并采用SuperCos1载体构建了绿木霉ZBS6黏粒基因组文库。试验中共获得3.9×104个黏粒克隆,平均插入片段大小约为33kb,文库滴度约为4.2×108 cfu/mL。理论上,从该文库中筛选到任一DNA序列的精确概率可高达99.9%。     

绿色木霉菌T-YY黄色素的稳定性研究

为开发一种新型的、功能性天然食品添加剂,该文以绿色木霉菌T-YY发酵液冻干粉(色素粗品)为试验材料,初步研究其紫外-可见光谱性质与稳定性.结果表明,色素粗品中至少含有4种以上不同性质的黄色素,该黄色素易溶于水和醇,色价高,在紫外区及可见光区各有一个吸收峰,最大吸收波长分别为286.5 nm和380 nm;在酸碱环境中色调保持亮黄色,其水溶液在pH＜5.0时出现浑浊,在pH≥5.0时呈亮黄色;pH值3.0～9.0色素溶液在室内自然光(25℃)下放置7 d后色调稳定;热稳定性好,100℃加热30 min色素保留率达到90.2%;抗H2O2、HNO3氧化能力强,但不耐Na2SO3还原作用.Fe3 、Cu2 和Mn2 的对色素具有消褪作用, Fe2 、Mg2 、Zn2 、Ca2 、K 、Na 则对其有不同程度的保护和增色作用.试验表明,绿色木霉T-YY黄色素在稳定性上呈现出一定优势,因此具有较大的开发潜力.     

绿色木霉代谢产物对黑曲霉和荔枝炭疽抑菌机理的研究

[目的]分析绿色木霉代谢产物对黑曲霉和荔枝炭疽抑菌机理。[方法]采用培养皿对峙培养,载片培养观察和透射电镜观察法分析黑曲霉和荔枝炭疽菌丝被绿色木霉代谢物拮抗后的变化。[结果]黑曲霉菌丝内部的细胞膜、线粒体、细胞核、细胞质等各种细胞器都已经消失或变形或干瘪;荔枝炭疽菌丝内部的液泡已经膨胀变形,线粒体和叶绿体经代谢物作用后分化成细小的颗粒,细胞核也变形,细胞质变成了胶体状态。[结论]黑曲霉和荔枝炭疽菌丝都不同程度变形,说明绿色木霉代谢物是一种重要的抑菌荆。     

Biological control of Rhizoctonia solani on potatoes by antagonists. 4. Inoculation of seed tubers with Verticillium biguttatum and other antagonists in field experiments

Inoculation of seed potatoes with the mycoparasiteVerticillium biguttatum, isolate M73 (combined withGliocladium roseum in 1981, either alone or mixed with isolate M180 plus antibiotics-producing isolates ofAzotobacter chroococcum in 1982) repeatedly proved successful in reducingRhizoctonia solani on stolons and stems. In field experiments, this ultimately led to a reduced formation of sclerotia on new tubers, particularly in neutral sandy loam and clay loam soils. In 1981 inoculation with antagonists led, when compared with no inoculation, to average reductions of 22 and 42% for the harvest from clean, and 15 and 26% for the harvest from infected seed tubers grown on slightly acid sandy soils and on neutral loam soils, respectively. The harvest from clean, inoculated seed tubers had the lowest sclerotium index. In 1982 inoculation of seed tubers planted in slightly acid sandy soils gave reductions of the sclerotium index of up to 22%. In the neutral marine loam soils considerable reductions were often achieved, viz., in slightly infected loams 51–68% and in rather heavily infected ones 4–43%. Chemical disinfection of seed tubers proved effective only in loam soils that were slightly infested withR. solani. In both years inoculation of seed tubers with antagonists led to significantly lower sclerotium indices of the harvest (p=0.1% in 1981; p=5% in 1982). V. biguttatum was present more frequently and in greater densities on stems and stolons of plants from inoculated than from non-inoculated seed tubers. The latter were colonized by wildV. biguttatum strains from the soil, apparently less effective antagonists.Early in the season, the soil temperature was too low for growth ofV. biguttatum. Nevertheless, inoculation of tubers that were planted early resulted in a considerable cotrol ofR. solani.Samenvatting Het beënten van poters met de opRhizoctonia solani parasiterende schimmelVerticillium biguttatum isolaat M73 in combinatie metGliocladium roseum (1981) of metV. biguttatum M73 alleen of in combinatie met isolaat M180 plus antibiotische isolaten van de bacterieAzotobacter chroococcum (1982), bleek effectief in het terugdringen of het onderdrukken vanR. solani op stengels en stolonen en het verminderen van de aantasting.Beënting van het pootgoed leidde tot een vermindering van de sclerotium (lakschurft)-vorming op de nieuwe knollen, vooral in klei-en zavelgronden. In 1981 leidde beënting van poters tot reductie in de sclerotiumvorming van gemiddeld 22 en 42% voor de oogst uit schoon en 15 en 26% voor de oogst uit besmet pootgoed geteeld op respectievelijk zandgrond en klei- en zavelgrond.In 1982 leidde beënten van de poters uitgeplant in licht zure zandgrond tot een gemiddelde reductie van de sclerotiumindex van de oogst van 22%. In zwaar besmette zandgrond trad evenwel geen reductie op; de infectiedruk was hier te groot. In de neutrale zavel- en kleigronden, vaak ook in de zwaarder besmette percelen werden aanzienlijke reducties bereikt, in de licht besmette gemiddeld 51–68% en in de zwaarder besmette 4–43%. Ontsmetten van pootgoed bleek alleen effectief in percelen die licht metR. solani waren besmet.In beide jaren bleek beënten van pootgoed met antagonisten te resulteren in een significant lagere sclerotiumindex van de oogst (p=0,1% in 1981; p=5% in 1982). V. biguttatum was veel vaker en meer aanwezig op de ondergrondse stengeldelen en stolonen van planten uit beënt pootgoed dan op die van niet beënte poters. De laatsten werden gekoloniseerd door wilde stammen vanV. biguttatum uit de grond, die vaak minder effectieve antagonisten waren. Beënting van vroeg gepote knollen — als de temperatuur nog te laag is voor de groei vanV. biguttatum — leverde toch gunstige resultaten op.     

Development ofClonostachys rosea and interactions withBotrytis cinerea in rose leaves and residues

The effect of microclimate variables on development ofClonostachys rosea and biocontrol ofBotrytis cinerea was investigated on rose leaves and crop residues. C.rosea established and sporulated abundantly on inoculated leaflets incubated for 7–35 days at 10°, 20° and 30°C and then placed on paraquat—chloramphenical agar (PCA) for 15 days at 20°C. On leaflets kept at 10°C, the sporulation after incubation on PCA increased from 60% to 93% on samples taken 7 to 21 days after inoculation, but decreased to 45% on material sampled after 35 days. A similar pattern was observed on leaves incubated at either 20° or 30°C. The sporulation ofC. rosea on leaf disks on PCA was not affected when the onset of high humidity occurred 0, 4, 8, 12 or 16 h after inoculation. However, sporulation was reduced to 54–58% on leaflets kept for 20–24 h under dry conditions after inoculation and before being placed on PCA. The fungus sporulated on 68–74% of the surface of leaf disks kept for up to 24 h at high humidity after inoculation, but decreased to 40–51% if the high humidity period before transferral to PCA was prolonged to 36–48 h. The growth ofC. rosea on leaflets was reduced at low inoculum concentrations (10³ and 10⁴ conidia/ml) because of competition with indigenous microorganisms, but at higher concentrations (10⁵ and 10⁶ conidia/ml) the indigenous fungi were inhibited. Regardless of the time of application ofC. rosea in relation toB. cinerea, the pathogen’s sporulation was reduced by more than 99%. The antagonist was able to parasitize hyphae and conidiophores ofB. cinerea in the leaf residues. AsC. rosea exhibited flexibility in association with rose leaves under a wide range of microclimatic conditions, and in reducingB. cinerea sporulation on rose leaves and residues, it can be expected to suppress the pathogen effectively in rose production systems.     

Metabolites of some biological control agents

The evidence is reviewed for the structure and biosynthesis of some metabolites of Trichoderma and Gliocladium species that are being considered as fungal biological control agents. The diverse biosynthetic capability of these organisms is proposed as a criterion for their potential application as bio-control agents.     

Suppression of sporulation ofBotrytis spp. as a valid biocontrol strategy

In this study, the hypothesis was tested that removal of substrate for sporulation ofBotrytis spp. may lead to a retardation of an epidemic if the majority of the inoculum is produced inside the treated crop. Suppression of sporulation ofBotrytis spp. could be an attractive option for biological control ofBotrytis leaf spot in onions. In a field experiment, necrotic leaf tissue was removed to simulate the effect of a biocontrol agent. By this means, the amount of substrate on whichBotrytis spp. sporulates was reduced. In the experiment, the spore load above the onion plots was significantly reduced and the epidemic of onion leaf spot was retarded. At the end of the growing season, the number of leaf lesions in the green leaf area was lower in plots with substrate removal than in control plots (0.6 and 1.1 cm^–2, respectively). The results demonstrated that an epidemic of onion leaf spot largely depends on the rate of inoculum production inside a crop. Thus, suppression of sporulation on necrotic leaf tissue is a valid control strategy that could be applied by using sporulation suppressing antagonists.     

绿色木霉L24对玉米幼苗生长的影响

采用绿色木霉L24菌株分生孢子可湿性粉剂浸种和叶面喷施两种方式处理玉米,通过对玉米幼苗的出苗率、根长、苗高、过氧化物酶含量、叶绿素含量、根系活力和丙二醛含量等形态指标和生理生化各项指标的测定,研究木霉L24菌株对玉米幼苗生长的影响。结果表明,木霉L24菌株分生孢子可湿性粉剂对玉米的出苗率、根长和苗高等均有促进作用,可有效提高玉米过氧化物酶含量、叶绿素含量和根系活力,降低丙二醛含量。     

绿色木霉Tr9701对多种病原菌的抑制作用及其抑病机理

对筛选获得的绿色木霉菌株Tr9701（Tr／ehodemmviride）进行抑菌活性测定。结果表明,Tr9701对立枯病菌和灰霉病菌等多种病原菌具有较强抑菌活性。小区试验表明,Tr9701菌剂对黄瓜立枯病的防治效果在66．27％-73．46％间,对番茄灰霉病的防病效果在70：85％-80．06％间。并通过产几丁质酶活性等对其抗病机理进行了初探,结果表明绿色木霉菌Tr9701产生几丁质酶,且经诱导处理酶产量明显提高．其酶粗提液对立枯病菌、灰霉病菌有显著抑制作用。显微观察显示绿色木霉菌对立枯病菌具有较强的寄生能力。     

Inoculum prevalence, host infection and biological control of Colletotrichum acutatum: causal agent of blueberry anthracnose in British Columbia

To identify the causal organism of anthracnose (ripe-rot), which reduces yield and postharvest quality of blueberries grown in British Columbia, Canada, 80 isolates were recovered from diseased fruits collected from commercial blueberry fields during 2002–04 and identified as Colletotrichum acutatum using colony morphology, growth rate and species-specific PCR primers. In vitro incubation of replicated sets of inoculated detached berries at various temperatures produced infection at temperatures of 7–30°C, with an optimum at 20°C. Colletotrichum acutatum could not survive on the soil surface as mummified berries but the pathogen was detected mostly within flower buds and less so in blueberry twigs and fruit trusses. Infection of developing flower buds in May–June of the preceding growing season gave the highest inoculum recovery in the following year. Two commercial fungal biocontrol agents, Prestop ( Gliocladium catenulatum ) and PlantShield ( Trichoderma harzianum ), each reduced anthracnose development in 2003 and 2004 by up to 45% when sprayed three times onto plants between flowering and fruit ripening.