The Role of Trichoderma harzianum Protease in the Biocontrol of Botrytis cinerea

The role of protease of Trichoderma harzianum in the biocontrol of Botrytis cinerea was examined. Two isolates of T. harzianum were compared for their ability to produce protease in liquid culture medium and on the surface of bean leaves. The biocontrol agent T. harziaum T39 produced 58mU/ml of protease and T. harzianum NCIM1185 produced 54mU/ml on the 5th day of growth in liquid culture medium. On bean leaves, combinations of B. cinerea and T. harzianum isolates were examined for the synthesis of protease. The protease activities were 0.9 and 0.6mU/ml for T. harzianum T39 and NCIM1185, respectively, and 0.5mU/ml for B. cinerea alone after 48h of incubation. In the presence of T. harzianum T39 culture liquid containing protease, a 55% reduction in B. cinerea germination and a 80% reduction in the germ tube length were observed after 17h of incubation in vitro. When T. harzianum isolates were added to B. cinerea on bean leaves, increased synthesis of protease was observed (1.0 and 1.2mU/ml for T39 and NCIM1185, respectively). In the presence of T. harzianum NCIM1185 protease, although the rate of germination was reduced, B. cinerea attained 98% germination after 17h of incubation. The hydrolytic enzymes produced by B. cinerea, endo-polygalacturonase (PG) and exoPG were partially deactivated by protease from the T. harzianum isolates. Carboxymethyl cellulase was deactivated only by protease of NCIM1185. On the surface of bean leaves, the protease (obtained from liquid culture medium of T. harzianum isolates) resulted in 56–100% reduction of disease severity. The culture liquid containing protease synthesized on the surface of bean leaves treated with B. cinerea and with T. harzianum was collected and added to fresh leaves infected by B. cinerea. There was 56–100% and 30–75% reduction of disease severity with liquid droplet collected from the leaves treated with T. harzianum T39 and NCIM1185, respectively. Increased control of disease was obtained by combining the conidia of T. harzianum isolates with protease obtained from culture media. Protease inhibitors, trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane (E64), antipain hydrochloride, and a mixture of inhibitors, but not pepstatin A, fully or partially nullified the biocontrol effect of T39. T39 was found to be a poor producer of chitinase and -1,3-glucanase in vitro. These enzymes were not detected on leaves treated with T39. Involvement of protease in biocontrol of B. cinerea is suggested.     

Differentiation of Trichoderma taxa associated with mushroom production

A major epidemic of green mould in mushroom compost in Northern Ireland lasting for 6 months was caused by a single taxon of Trichoderma harzianum (Th2). This has continued to be the principal taxon causing losses in Ireland over a 9-year period. A separate taxon, Th4 , is responsible for a more recent epidemic in North America. Cultural methods are described which aid rapid differentiation of four distinct taxa of T. harzianum and five other species from the mushroom environment. After developing a standard cultural method, isolates were grouped visually by features such as growth rate at 27°C, amount of aerial mycelium, and the effect of light on sporulation form and timing. Most of these groups of isolates had significantly different growth rate ratios from each other at 27°C/17°C. Furthermore isolates within groups which had been separated by culture features alone were closely homologous as regards their microscopic morphological features such as phialides and phialospores. Two factors underlining the importance of using a standard cultural method in aiding identification were that culture morphology was shown to vary widely on different media and spore size was found to vary significantly with incubation temperature. Some difficulties are discussed concerning differentiation of Trichoderma spp. using classical microscopic features alone.     

A mini-bag technique for evaluation of fungicide effects on Trichoderma spp in mushroom compost

An in vivo technique was developed to observe colonisation of mushroom compost by Trichoderma spp. Isolates of T. harzianum (Th2), T. harzianum (Th1), T. koningii (Tk) and T. viride (Tv) were artificially introduced into compost using a mini-bag system. Wheat grains, colonised by Trichoderma spp, were placed centrally on a layer of compost at the bottom of 1-litre polythene bags which were then filled with 350 g of spawned or un-spawned compost, and partially sealed. After 14 and 21 days incubation at 27 degrees C, the bags were assessed for recovery of Trichoderma from middle and top zones using a needle stab re-isolation technique and a visual colonisation scoring system. Visible green mould contamination, similar to that observed in practice, developed within 21 days. The visual colonisation scoring was reliably related to the re-isolation success. In this evaluation, Trichoderma spp showed considerable differences in their relative abilities to colonise spawned and un-spawned compost, with Th2 isolates being consistently superior to the other isolates of Th1, Tk and Tv in colonising spawned compost. This technique was employed to evaluate the effects of fungicides on the colonisation of mushroom compost by three Trichoderma spp: Th2, Th1 and Tk, using 1-litre and 5-litre mini-bag systems. Aqueous suspensions of benomyl, carbendazim, thiabendazole, prochloraz and prochloraz+carbendazim incorporated into the compost at 50 mg litre(-1), or applied to spawn at 50 mg kg(-1), reduced the colonisation by Trichoderma spp. Prochloraz and prochloraz+carbendazim were superior to benomyl, carbendazim or thiabendazole in reducing compost colonisation by Th2, Th1 and Tk, with Th2 being the most persistent type, capable of colonising treated compost in the presence of all five fungicides. The prochloraz+carbendazim mixture, not normally used in mushroom production, was equal to or better than prochloraz alone. The incidence of green mould colonisation by Th2 was as extensive in the 5-litre compost bags as in the 1-litre bags, but colonisation by Th1 and Tk was more apparent in the 5-litre bags. The in vivo mini-bag evaluations using wheat grain Trichoderma inoculum and needle stab re-isolation procedures proved an efficient method for studying colonisation and screening for effectiveness of fungicides applied to mushroom compost or spawn.     

木霉对草坪褐斑病的拮抗效果及耐药性

通过木霉属5菌株与大连高尔夫球场上草坪褐斑病菌的对峙培养试验,研究结果表明:哈茨木霉Thar1菌株、哈茨木霉Thar2菌株、深绿木霉Tat菌株、钩状木霉Tha菌株及桔绿木霉Tci菌株对病原菌的抑制率为:66%、73%、71%、77%、55%,而相对抑菌效果分别为2.06、3.94、2.35、3.54、2.27,可以作为草坪褐斑病菌的生防菌加以利用。在这5株木霉中以哈茨木霉Thar2对草坪褐斑病菌的拮抗作用最强。观察结果表明,哈茨木霉Thar2对草坪褐斑病菌的拮抗机制主要表现为生长竞争、重寄生及产生抗菌物质。     

Green Mold Diseases of Agaricus and Pleurotus spp. Are Caused by Related but Phylogenetically Different Trichoderma Species

ABSTRACT Producers of champignon (Agaricus bisporus) and oyster mushroom (Pleurotus ostreatus) are facing recent incidents of green mold epidemics in Hungary. We examined 66 Trichoderma strains isolated from Agaricus compost and Pleurotus substrate samples from three Hungarian mushroom producing companies by a polymerase chain reaction-based diagnostic test for T. aggressivum, sequence analysis of the internal transcribed spacer region 1 (ITS1) and ITS2 and (selectively) of the fourth and fifth intron of translation elongation factor 1alpha (tef1alpha), and restriction fragment length polymorphism of mitochondrial DNA. Seven Trichoderma species were identified: T. aggressivum f. europaeum (17 isolates), T. harzianum (three isolates), T. longibrachiatum (four isolates), T. ghanense (one isolate), T. asperellum (four isolates), T. atroviride (nine isolates), and a still undescribed phylogenetic species, Trichoderma sp. DAOM 175924 (28 isolates). T. aggressivum f. europaeum was exclusively derived from A. bisporus compost, whereas Trichoderma sp. DAOM 175924 exclusively occurred in the substrate for Pleurotus cultivation. Sequences of the latter strains were co-specific with those for Trichoderma pathogens of P. ostreatus in Korea. The widespread occurrence of this new species raises questions as to why infections by it have just only recently been observed. Our data document that (i) green mold disease by T. aggressivum f. europaeum has geographically expanded to Central Europe; (ii) the green mold disease of P. ostreatus in Hungary is due to the same Trichoderma species as in Korea and the worldwide distribution of the new species indicates the possibility of spreading epidemics; and (iii) on mushroom farms, the two species are specialized on their different substrates.     

Induced systemic resistance in Trichoderma harzianum T39 biocontrol of Botrytis cinerea

Biocontrol of Botrytis cinerea with Trichoderma spp. is generally believed to result from direct interaction of the biocontrol agent with the pathogen or from a Trichoderma-induced change in environmental conditions that affects B. cinerea development. In this work we provide arguments for the participation of induced plant defence in T. harzianum T39 control of B. cinerea. In tomato, lettuce, pepper, bean and tobacco, T. harzianum T39 application at sites spatially separated from the B. cinerea inoculation resulted in a 25–100%percnt; reduction of grey mould symptoms, caused by a delay or suppression of spreading lesion formation. Given the spatial separation of both micro-organisms, this effect was attributed to the induction of systemic resistance by T. harzianum T39. The observation that in bean the effect of T. harzianum T39 was similar to that of the rhizobacterium Pseudomonas aeruginosa KMPCH, a reference strain for the induction of systemic resistance, confirmed this hypothesis. Since B. cinerea control on tobacco leaves sprayed with T. harzianum T39 was similar to the control on leaves from T. harzianum T39 soil-treated plants, induction of plant defence might also participate in biocontrol on treated leaves.     

生防木霉菌Th2和T4对甜瓜根围土壤微生态的影响

利用平板计数、微生物量碳和末端标记限制性片段长度多态性分析(terminal restriction fragment length polymorphism,T-RFLP)相结合的方法研究了2株生防菌哈茨木霉Th2和T4对甜瓜根围土壤微生态的影响.结果表明,生防菌Th2和T4对根围土壤细菌有明显的促生作用,而对真菌和放线菌有明显的抑制作用.T-RFLP数据统计学分析表明,Th2和T4的引入可以增加土壤细菌的多样性.其中对开花期土壤细菌菌群影响最显著,Th2对TRF251和TRF513菌群有显著的促生作用,含量是对照的3.7倍和4.5倍,T4对TRF251和TRF513菌群含量也比对照高4倍和5.6倍,而2株生防菌对TRF60菌群均有显著的抑制作用,其含量分别为对照的51.6%和64.3%.在果熟期生防菌对土壤细菌菌群的影响减弱,表明土壤的缓冲功能发挥了作用.研究结果还表明,Th2和T4对土壤细菌菌群有着相似的促生和抑制作用,且不会破坏土壤微生态的稳定性.     

哈茨木霉SH2303防治玉米小斑病的初步研究

哈茨木霉SH2303是本实验室分离获得的1株具有生防效果的菌株,该菌株能够较好的防治玉米小斑病。通过离体叶片试验确定哈茨木霉SH2303诱导玉米抗小斑病的持效期达15 d。盆栽及大田试验表明,防治玉米小斑病防效分别达到78.1%和56.3%。盆栽试验表明,哈茨木霉 SH2303处理的叶片在挑战接种小斑病菌后第36 h,玉米体内防御反应酶系PAL和SOD活性达到峰值。同时,防御基因Pal和Sod的表达水平也明显上升。综合分析表明,哈茨木霉SH2303的诱导抗性作用是防治玉米小斑病的重要机制之一。     

Dynamics of soilborne Rhizoctonia solani in the presence of Trichoderma harzianum: effects on stem canker, black scurf and progeny tubers of potato

Stem canker and black scurf are diseases of potato caused by the fungus Rhizoctonia solani . Spatiotemporal experimentation and empirical modelling were applied for the first time to investigate the effect of antagonistic Trichoderma harzianum on the dynamics of soilborne R. solani on individual potato plants. Trichoderma harzianum reduced the severity of symptoms, expressed as 'rhizoctonia stem lesion index' (RSI), during the first 7 days post-inoculation when the inoculum of R. solani was placed at certain distances (30–60 mm) from the host. For example, with inoculum at 40 mm from the host, RSI was 6 and 40 with and without T. harzianum , respectively. At later observation times, the antagonistic effect was overcome. Trichoderma harzianum reduced the severity of black scurf on progeny tubers. Furthermore, the mean number of progeny tubers per potato plant was reduced by the biocontrol treatment (means of 6·5 ± 1·1 and 9·9 ± 2·7 tubers per plant with and without T. harzianum , respectively), as was the proportion of small (0·1–20·0 g) tubers (48% and 66% with and without T. harzianum , respectively). Additionally, there were fewer malformed and green-coloured tubers in pots treated with T. harzianum than in those without T. harzianum .     

Evaluation of Trichoderma spp. as a Biocontrol Agent Against Soilborne Fungi and Plant-parasitic Nematodes in Israel

The optimal conditions required to market Trichoderma as a biocontrol agent against soilborne fungi and nematodes are discussed. These include a proper formulation, an efficient delivery system, and alternative methods for Trichoderma's application.The implementation of Trichoderma in integrated pest management (IPM) can be achieved using a soil treatment which combines reduced amounts of biocides/fungicides and the Trichoderma preparation. Biocontrol activity can be increased by combining two (or more) types of biocontrol agents. Moreover, the construction of a genetically modified Trichoderma can lead to the improvement of certain traits which are absent or not highly expressed in the native microorganism isolated from its natural habitat.Different Trichoderma harzianum and T. lignorum isolates were tested for their nematicidal activity against the root-knot nematode Meloidogyne javanica. In short-term experiments, improved growth of nematode-infected plants and decreases in the root-galling index and the number of eggs per gram of root were achieved when nematode-infested soils were pre- exposed to the T. harzianum preparations. A long-term experiment resulted in improved growth and higher yield of nematode-infected plants, but no significant change in the galling index, either by pre-exposure of the fungus to the soil or by enrichment in the root-ball.As biocontrol is an integral part of the IPM philosophy, judicious use of Trichoderma against soilborne pathogens, when demonstrated to be consistently effective, practical and economic, can serve as a model for the introduction and implementation of other biocontrol means into IPM.     

哈茨木霉对接种尖孢镰刀茵后黄瓜根系次生代谢物的影响

采用室内筛选与田间试验相结合的方法,对哈茨木霉抑制黄瓜枯萎病菌的拮抗机制进行了研究.对峙培养结果显示木霉菌和病原菌之间形成了较明显的抑菌圈,其中菌株TN对枯萎病菌抑制作用较强.接种木霉菌植株显著提高了植株中肉桂醇脱氢酶（CAD）、多酚氧化酶（PPO）、愈创木酚过氧化物酶（G-POD）、咖啡酸过氧化物酶（CA-POD）和绿原酸过氧化物酶（CGA-POD）的活性及总酚、类黄酮、木质素的含量.接种木霉菌可诱导根系次生代谢相关基因的上调表达,C4H、CAD、CCOMT、G6PDH、PAL和PR-1的表达量分别为对照的 5.64、5.31、4.28、15.33、7.36 和 6.45 倍.表明木霉菌诱导的黄瓜根部对枯萎病的抗性与植物次生代谢密切相关.     

Use of Trichoderma harzianum in combination or alternation with fungicides to control cucumber grey mould (Botrytis cinerea) under commercial greenhouse conditions

A preparation of Trichoderma harzianum was sprayed on cucumber plants in greenhouses in order to control fruit and stem grey mould. Up to 90% control was achieved by the biocontrol agent (0·5–1·0 g/l) which in most experiments under commercial conditions was as effective as the dicarboximide fungicides iprodione or vinclozolin (0·5 g/l each) alone or alternated with diethofencarb + carbendazim (0·25 g/l each). However, in one experiment disease incidence in Trichoderma -treated plots did not differ significantly from the control. A mixture of T. harzianum with a dicarboximide fungicide resulted in up to 96% control of grey mould. In this case control was always significant ( P =0·05) but improvement of control compared with each treatment alone was not significant ( P =0·05). The alternation of sprays with the biocontrol preparation and with a dicarboximide fungicide was tested in three out of the five experiments and was found to be effective, thus enabling a reduction in the use of chemical sprays. Populations of T. harzianum were on a level of 3 × 10⁵-8 × 10⁵ c.f.u. per leaf and ten times lower on one fruit. They remained high after the second and third sprays. Conditions favouring the ability of T. harzianum to control grey mould were temperatures above 20°C and relative humidity between 80 and 97%.     

Effect of Gliocladium and Trichoderma on damping-off and blight of snapbean caused by Sclerotium rolfsii in the greenhouse

Aqueous suspensions of conidia of 285 wild-type strains and mutants of Gliocladium virens, Trichoderma hamatum, T. harzianum and T. viride were tested against Sclerotium rolfsii in the greenhouse. Ten strains of G. virens and four strains of T. harzianum suppressed damping-off of snapbean by 30-50% and blight by 36-74%. All strains of T. hamatum and T. viride tested as conidia were ineffective. In general, strains of G. virens were more effective in suppressing disease in the greenhouse than strains of T. harzianum. Several strains of G. virens and T. harzianum used alone were equal to or more effective than double and triple mixtures of such strains in disease suppression. Of four formulations of G. virens tested, germlings, alginate-bran-fermenter biomass pellets, and Pyrax-fermenter biomass mixtures reduced disease considerably and all three formulations were more effective than conidia in aqueous suspension. Strain G1-3 of G. virens added to soil as Pyrax-fermenter biomass mixtures in amounts to provide colony-forming units ranging from 1 -5 × 10³ to 1 -2 × 10⁴ per g soil provided statistically significant protection of the host at all concentrations. The extent of biological control with strains G1-3 and G1-21 of G. virens also depended on the strain of the pathogen used. Both strains effectively suppressed disease caused by strain Sr-1 (small sclerotia) of S. rolfsii. They were partially effective against strain Sr-116 (medium size sclerotia) and ineffective against strain Sr-3 (large sclerotia). Although strains G1-3 and G1-21 colonized the sclerotia of all three strains of S. rolfsii in soil effectively, they only reduced germinability of sclerotia of strain Sr-1.     

Incorporation of Weather Forecasting in Integrated, Biological-Chemical Management of Botrytis cinerea

ABSTRACT A strategy for integrated biological and chemical control of Botrytis cinerea in nonheated greenhouse vegetables was developed. The biocontrol agent used was a commercial preparation developed from an isolate of Trichoderma harzianum, T39 (Trichodex). Decisions concerning whether to spray the biocontrol agent or a fungicide were made based on a weather-based disease warning system. The integrated strategy (BOTMAN [short for Botrytis manager]) was implemented as follows: when slow or no disease progress was expected, no spraying was needed; when an outbreak of epidemics was expected, use of a chemical fungicide was recommended; in all other cases, application of T. harzianum T39 was recommended. Future weather information (a 4-day weather forecast provided by the Israel Weather Forecast Service) was more useful for disease warnings than immediate past weather. The integrated strategy was compared with weekly applications of fungicide in 11 experiments conducted over 3 years in greenhouse-grown tomato and cucumber. Disease reduction in the integrated strategy (63.9 +/- 3.0%) did not differ significantly (P < 0.05) from the fungicide-only treatment (70.1 +/- 3.6%). The number of fungicide sprays in the integrated strategy ranged from 2 to 7 (mean 4.2) compared to 7 to 13 (mean 10.5) in the fungicide treatment. The integrated strategy averaged 5.9 sprays of T. harzianum T39. For the integrated strategy, one treatment omitted use of T. harzianum T39 to estimate the contribution of this agent to disease control. Disease reduction in that treatment (49.1 +/- 4.8%) was significantly (P < 0.05) inferior to the combined chemical and biological strategy, indicating that the T. harzianum T39 sprays had a measurable effect on disease control.     

Ecological Studies of Transformed Trichoderma harzianum Strain 1295-22 in the Rhizosphere and on the Phylloplane of Creeping Bentgrass

ABSTRACT A beta-glucuronidase (GUS) reporter gene and a hygromycin B (hygB) phosphotransferase gene were integrated separately into the Trichoderma harzianum strain 1295-22 genome, using biolistic transformation. The mycelial growth and biocontrol ability of the transformed strains did not differ from that of the original strain. The transformed Gus(+)-kanamycin-resistant (Gus(+)Kan(R)) strains were used to monitor growth and interactions with Rhizoctonia solani on creeping bentgrass plants. The hygB-resistant (hygB(R)) strains were used to selectively recover strain 1295-22 from the rhizosphere soil and phylloplane of creeping bentgrass after spray applications. The population levels of two hygB(R) strains and the original strain were very similar for all treatments. All three strains persisted for the duration of the experiment (28 days) in both the rhizosphere soil and on leaves, although population levels declined somewhat over the course of the experiment in unautoclaved soils. In this study, the results demonstrated that hygB(R) strains remained dominant over time when assayed on Trichoderma-selective medium containing hygB. The hygB(R) strains were not displaced by strains that colonized untreated plants. Microscopic observation showed that the Gus(+)Kan(R) strains colonized the rhizoplane, seed coat, and phylloplane of creeping bentgrass. These results supported our earlier observation that strain 1295-22 was rhizosphere and phyllo-plane competent. Interactions between T. harzianum and R. solani were readily observed in situ and changed over time. Two types of reactions were found in these experiments. In the first type, sections of hyphae of R. solani near the hyphae of T. harzianum appeared damaged, and the pathogen appeared necrotic when viewed with a microscope. The second type, observed less frequently than the first type, was typical of myco-parasitism. The findings in this study provide new insight into the interactions between R. solani and T. harzianum, providing a basis for future research.     

Application of Trichoderma and Gliocladium in alginate pellets for control of Rhizoctonia damping-off

Alginate pellets were prepared from wet fermentor biomass of 11 isolates of Trichoderma spp. and Gliocladium virens , with wheat bran as a food base carrier. Pellets with eight of the isolates reduced survival (34-78%) of Rhizoctonia solani in infested beet seed in soil. Pellets containing a T. harzianum isolate (Th-58) and a T. hamatum isolate (TRI-4) were the most effective. All isolates significantly reduced growth of the pathogen from infested beet seed into natural soil. Populations of isolates proliferated in soil to 10⁶−10¹¹ colony-forming units/g (cfu) from propagules within the pellets. Pellets with TRI-4 reduced pathogen survival and growth (>70%) in six different soils and were effective against six R. solani isolates in a natural loamy sand. Survival of R. solani in infested beet seed was not reduced when TRI-4 pellets were added to soil 1-6 weeks before the pathogen; however, saprophytic growth was prevented. Small amounts of biomass (3.0–7.5 g wet weight) in pellets were as effective as a large amount (300 g) in suppressing the pathogen. The storage of pellets for more than 6 weeks at 5 or 25C reduced their effectiveness against R. solani. Pellets prepared with four and three of the 11 isolates prevented damping-off of cotton and sugar beet in the greenhouse, respectively.     

Influence of a Fungus-Feeding Nematode on Growth and Biocontrol Efficacy of Trichoderma harzianum

ABSTRACT A fungivorous nematode, Aphelenchoides sp., was isolated from field soil by baiting with mycelium of the biocontrol fungus Trichoderma harzianum ThzID1, and subsequently was maintained on agar cultures of the fungus. Interactions between the nematode and the green fluorescent protein-producing transformant, T. harzianum ThzID1-M3, were investigated in both heat-treated (80 degrees C, 30 min) and untreated field soil. ThzID1-M3 was identified in soil by epifluorescence microscopy. When ThzID1-M3 was added to soil as an alginate pellet formulation, addition of the nematode (10 per gram of soil) significantly reduced radial growth and recoverable populations of the fungus, and the effect was greater in heat-treated soil than in untreated soil. Addition of ThzID1-M3 to soil pretreated with the nematode (10 per gram of soil) stimulated nematode population growth for approximately 10 to 20 days, whereas nematode populations decreased in the absence of added Trichoderma sp. When sclerotia of Sclerotinia sclerotiorum were added to soil (10 per 200 g of soil) with ThzID1-M3 (40 pellets per 200 g of soil), addition of Aphe-lenchoides sp. (2,000 per 200 g of soil) reduced the number of sclerotia colonized by ThzID1-M3. These results suggest that fungivorous nematodes may be a significant biotic constraint on activity of biocontrol fungi in the field.     

哈茨木霉几丁质酶诱导及其对水稻纹枯病菌的拮抗作用

 在实验室条件下分别以几丁质和水稻纹枯病菌(Rhizoctonia solani)细胞壁作唯一碳源诱导哈茨木霉(Trichoderma harzianum)菌株NF9、TC3和P1产生几丁质酶,用硫酸铵沉淀法制备几丁质酶粗提液。上述木霉菌株内切几丁质酶活性(对胶体几丁质浑浊度的减少率)分别为79.8%、74.4%和76.0%,均显著高于非诱导的阳性对照。培养第5 d几丁质诱导的木霉菌株NF9和TC3内切几丁质酶活性显著高于由水稻纹枯病菌细胞壁诱导的酶活性。体外测定表明,通过诱导的木霉菌株TC3、NF9和P1几丁质酶粗提液对水稻纹枯病病菌的拮抗圈直径可达38、21和23 mm,与非诱导的阳性对照比较有显著性差异。对木霉几丁质酶拮抗作用的特点及生防应用进行了讨论。     

Diversity of soil-dwelling Trichoderma in Colombia and their potential as biocontrol agents against the phytopathogenic fungus Sclerotinia sclerotiorum (Lib.) de Bary

Twenty-one isolates of Trichoderma spp. were collected from eight states in Colombia and characterized based on the 5′ end of the translation elongation factor-1α (EF1-α1) gene and RNA polymerase II gene encoding the second largest protein subunit (RPB2) by using mixed primers. Seven species of soil-dwelling Trichoderma were found: T. atroviride, T. koningiopsis, T. asperellum, T. spirale, T. harzianum, T. brevicompactum and T. longibrachiatum. Species identifications based on the EF1-α1 gene were consistent with those obtained from the RPB2 gene. Phylogenetic analyses with high bootstrap values supported the validity of the identification of all isolates. These results suggest that using the combination of the genes EF1-α1 and RPB2 is highly reliable for molecular characterization of Trichoderma species. Trichoderma asperellum Th034, T. atroviride Th002 and T. harzianum Th203 prevented germination of more than 70 % of sclerotia of Sclerotinia sclerotiorum in bioassay tests and are promising biological control agents. No relationship between mycelium growth rate and parasitism level was found.     

Identification of Armillaria isolates from Bhutan based on DNA sequence comparisons

Armillaria root rot is a serious disease in fir and mixed conifer forests of Bhutan, Eastern Himalayas. The species causing this disease have, however, never been identified. The aim of this study was to identify field isolates collected at four localities in Bhutan. Identification was based on RFLP analysis of the IGS-1 region, comparisons of ITS and IGS-1 sequence data with those available on GenBank, cladistic analyses and sexual compatibility studies. Isolates were found to reside in two distinct RFLP groups. RFLP group 1 isolates from Pinus wallichiana at Yusipang had RFLP profiles and IGS-1 sequences similar to those of Armillaria mellea ssp. nipponica . Although ITS sequence data are not available for A. mellea ssp. nipponica , sequences from this DNA region were most similar to the closely related A. mellea from Asia. The RFLP profile and IGS-1 sequences for RFLP group 2 isolates from Abies densa at Changaphug, Tsuga dumosa at Chimithanka as well as Picea spinulosa and T. dumosa in the Phobjikha valley were similar to those published for Armillaria borealis , Armillaria cepistipes , Armillaria gemina and Armillaria ostoyae . Distance analysis based on IGS-1 and ITS sequence data indicated that these isolates are closely related to A. cepistipes , Armillaria gallica and Armillaria sinapina . The isolates were, however, sexually incompatible with tester strains of A. cepistipes , A. gallica and A. sinapina . Although closely related to these species, they appear to represent a distinct taxon that will be referred to as Bhutanese phylogenetic species I (BPS I) until basidiocarps are found and the species can be described.