Trichoderma spp. and Bacillus spp. as growth promoters in maize (Zea mays L.)

Microbes that are beneficial to plants are used to enhance the crop growth, yield and are alternatives to chemical fertilizers. Trichoderma and Bacillus are the predominant plant growth-promoting fungi and bacteria. The objective of this study was select, characterize, and evaluate isolates of Trichoderma spp. and Bacillus spp. native from the northern region of Sinaloa, Mexico, and assess their effect on growth promotion in maize (Zea mays L.). In greenhouse conditions, four Trichoderma isolates and twenty Bacillus isolates, as well as two controls, were tested in a completely randomized design with three replicates. We selected the two best strains of Trichoderma and Bacillus: TB = Trichoderma asperellum, TF = Trichoderma virens, B14 = Bacillus cereus sensu lato and B17 = Bacillus cereus, which were evaluated in the field in a completely randomized blocks in factorial arrangement design with three replicates applying different rates of nitrogen fertilizer (0, 150 kg N/ha, and 300 kg N/ha). Treatments 5 (B17 = B. cereus) and 11 (TF = T. virens) both fertilized with 150 kg N/ha showed similar yields and they did not reveal significant differences from the treatments fertilized with 300 kg N/ha. This indicated that treatment 5 (B17= B. cereus with 150 kg N/ha) and treatment 11 (TF= T. virens with 150 kg N/ha) were efficient as growth promoters, by not showing significant differences in root volume and dry weight of foliage. The results indicated a reduction of 50% in the rate of nitrogen to fertilizer required for maize (Zea mays L.) crops. These microorganisms Trichoderma and Bacillus could be an alternative to reduce the use of chemical fertilizers in maize.     

Biological control of cucumber and sugar beet damping-off caused by Pythium ultimum with bacterial and fungal antagonists

AIMS: Five bacterial strains belonging to Bacillus subtilis, Pseudomonas fluorescens and Ps. corrugata and two fungal strains belonging to Trichoderma viride and Gliocladium virens were evaluated for their efficacy in controlling sugar beet and cucumber damping-off caused by Pythium ultimum. METHODS AND RESULTS: The in vitro antagonistic activity of bacteria against various Pythium spp. was evaluated with dual cultures in various media. Pseudomonas strains inhibited the pathogen better than Bacillus strains. To identify potentially useful antagonist combinations, dual compatibility of antagonists was also evaluated, based on growth in two liquid media containing substrate previously used by other antagonists. Four pairs of bacteria were selected. Sugar beet damping-off biocontrol was attempted with bacterial seed treatments (individually and in pairs). Cucumber damping-off biocontrol was attempted with bacterial seed treatments and bacterial and fungal compost treatments. In sugar beet, satisfactory biocontrol was only achieved with Pseudomonas antagonists. Antagonist combinations did not show any superior biocontrol ability to individual antagonists and compatibility of bacteria in vitro did not correlate with compatibility in vivo. Bacterial seed treatments and fungal compost treatments failed to control cucumber damping-off. Better biocontrol in cucumber was achieved when bacterial antagonists were applied by drenching or by coating seed with bacteria in a peat carrier. CONCLUSIONS: Pseudomonas antagonists were superior to Bacillus antagonists in controlling damping-off in cucumber and sugar beet. Pseudomonas peat inocula maintained a good shelf-life 2 years after preparation. SIGNIFICANCE AND IMPACT OF THE STUDY: Pseudomonas peat formulations have the potential for development into commercial biopesticides.     

Genetic and metabolic diversity of Trichoderma: a case study on South-East Asian isolates

We have used isolates of Trichoderma spp. collected in South-East Asia, including Taiwan and Western Indonesia, to assess the genetic and metabolic diversity of endemic species of Trichoderma. Ninety-six strains were isolated in total, and identified at the species level by analysis of morphological and biochemical characters (Biolog system), and by sequence analysis of their internal transcribed spacer regions 1 and 2 (ITS1 and 2) of the rDNA cluster, using ex-type strains and taxonomically established isolates of Trichoderma as reference. Seventy-eight isolates were positively identified as Trichoderma harzianum/Trichoderma inhamatum (37 strains) Trichoderma virens (16 strains), Trichoderma spirale (8 strains), Trichoderma koningii (3 strains), Trichoderma atroviride (3 strains), Trichoderma asperellum (4 strains), Hypocrea jecorina (anamorph: Trichoderma reesei; 2 strains), Trichoderma viride (2 strains), Trichoderma hamatum (1 strain), and Trichoderma ghanense (1 strain). Analysis of biochemical characters revealed that T. virens, T. spirale, T. asperellum, T. koningii, H. jecorina, and T. ghanense formed clearly defined clusters, thus exhibiting species-specific metabolic properties. In biochemical character analysis T. atroviride and T. viride formed partially overlapping clusters, indicating that these two species may share overlapping metabolic characteristics. This behavior was even more striking with T. harzianum/T. inhamatum where genotypes defined on the basis of ITS1 and 2 sequences overlapped significantly with adjacent genotypes in the biochemical character analysis, and four strains from the same location (Bali, Indonesia) even clustered with species from section Longibrachiatum. The data indicate that the T. harzianum/T. inhamatum group represents species with high metabolic diversity and partially unique metabolic characteristics. Nineteen strains yielded three different ITS1/2 sequence types which were not alignable with any known species. They were also uniquely characterized by morphological and biochemical characters and therefore represent three new taxa of Trichoderma.     

Variation of Fungicide Resistance in Czech Populations of Pseudoperonospora cubensis

During the growing seasons between the years 2001 and 2004, 98 isolates of Pseudoperonospora cubensis from nine regions of Czech Republic were collected and screened for tolerance/resistance to the three frequently used fungicides (propamocarb, fosetyl‐Al, metalaxyl). Fungicides were tested in five different concentrations, using a floating disc bioassay. Fungicide effectiveness varied considerably. Propamocarb appeared most effective and all the isolates collected in the years 2001–2003 were found sensitive to all tested concentrations [607–9712 μg active ingredient (a.i.)/ml]. In 2004, some strains with increased resistance to propamocarb were detected. These strains were characterized by tolerance at the lowest concentrations (607 μg a.i./ml, eventually on 1214 μg a.i./ml); however, they were controlled by 2428 μg a.i./ml. Fosetyl‐Al was effective at the recommended concentration of 1600 μg a.i./ml against all isolates. However, the occurrence of isolates (collected in 2001) which sporulated at low concentrations (400 and 800 μg a.i./ml) indicated that the selection for tolerance occurs in the pathogen population. Nevertheless, this phenomenon was not confirmed with the P. cubensis isolates collected between the years 2002 and 2004. Metalaxyl was found ineffective, because 97% of the isolates showed the resistance to the recommended concentration (200 μg a.i./ml), and the other 3% of isolates expressed tolerant response. The majority of the isolates showed profuse and/or limited sporulation at higher concentrations (400 and 800 μg a.i./ml). A substantial shift to highly metalaxyl resistant strains was evident in the Czech P. cubensis populations during 2001–2004.     

木霉菌在玉米病害生物防治中的作用机制及应用

目前,世界上共有分属于10个属（Trichoderma,Gliocladium,Chaetomium,Bacillus,Burkhoderia,Streptomyces and Pseudomonas,Pantoea,Enterobacter,Macrobacterium）中的微生物被试验用于玉米病害的生物防治,其中细菌14种,真菌17种,放线菌1种。国际上由木霉菌开发的生物杀菌剂和生物肥料有50余种,其中以哈茨木霉T22菌株开发的产品最为著名。目前在我国也开发出了4种木霉菌剂型（可湿性粉剂、颗粒剂、水分散粒剂和种衣剂）,正式登记的木霉菌杀菌剂有7种,其中6种为可湿粉剂,1种为水分散粒剂,主要登记用于防治番茄、观赏百合、黄瓜的立枯病、猝倒病、根腐病、灰霉病、霜霉病以及小麦的纹枯病,但尚无木霉菌生物农药被登记用于防治玉米病害。以木霉菌为主要成分登记的菌肥产品有11种,其中在玉米上应用的有2种。由课题组研制的木霉菌颗粒剂和种衣剂通过土壤穴施和种子包衣可有效防治玉米茎腐病和纹枯病,其中木霉菌颗粒剂防效达65%-87%。近期研制的木霉菌可湿性粉剂对玉米小斑病的防效达50%-60%。国际上已鉴定出多种可诱导玉米获得系统抗性的木霉菌源激发子,其中包括Sm1、纤维素酶、疏水蛋白和Avr4 /Avr9等效应因子。本课题组近年鉴定出Thc6（锌指蛋白类转录因子）、PAF-AH和Thph1/Thph2的编码产物在系统诱导以JA/ET信号调控的玉米抗弯孢菌叶斑病中具有重要作用,符合植物免疫MAMPs模式,为全面认识木霉菌诱导免疫机理提供了重要理论依据。木霉菌诱导玉米从根至叶片的防御反应系统传导机制还需深入研究。     

Effect of fungicides,insecticides and allosamidin on a thermostable chitinase from Bacillus sp. BG-11

A purified, thermostable chitinase from Bacillus sp. BG-11 retained 90% of its activity in the presence of fungicides such as Aromex, Captafol, Captan, Chlorothalonil, Dinocap, Metalaxyl, sulphur, Triadimefon, and insecticides such as Acephate, Chloropyriphos, Cypermethrin, Diclovorus, Dimethoate, Methomyl, Malathion, Methylparathion and Monocrotophos at a concentration of 100 g active ingredient per ml of enzyme solution. Allosamidin inhibited the chitinase with IC₅₀ value of 48 M.     

Biochemical and metabolic profiles of Trichoderma strains isolated from common bean crops in the Brazilian Cerrado, and potential antagonism against Sclerotinia sclerotiorum

Some species of Trichoderma have successfully been used in the commercial biological control of fungal pathogens, e.g., Sclerotinia sclerotiorum, an economically important pathogen of common beans (Phaseolus vulgaris L.). The objectives of the present study were (1) to provide molecular characterization of Trichoderma strains isolated from the Brazilian Cerrado; (2) to assess the metabolic profile of each strain by means of Biolog FF Microplates; and (3) to evaluate the ability of each strain to antagonize S. sclerotiorum via the production of cell wall-degrading enzymes (CWDEs), volatile antibiotics, and dual-culture tests. Among 21 isolates, we identified 42.86% as Trichoderma asperellum, 33.33% as Trichoderma harzianum, 14.29% as Trichoderma tomentosum, 4.76% as Trichoderma koningiopsis, and 4.76% as Trichoderma erinaceum. Trichoderma asperellum showed the highest CWDE activity. However, no species secreted a specific group of CWDEs. Trichoderma asperellum 364/01, T. asperellum 483/02, and T. asperellum 356/02 exhibited high and medium specific activities for key enzymes in the mycoparasitic process, but a low capacity for antagonism. We observed no significant correlation between CWDE and antagonism, or between metabolic profile and antagonism. The diversity of Trichoderma species, and in particular of T. harzianum, was clearly reflected in their metabolic profiles. Our findings indicate that the selection of Trichoderma candidates for biological control should be based primarily on the environmental fitness of competitive isolates and the target pathogen.     

马铃薯晚疫病生防木霉菌的筛选及鉴定

采用马铃薯活体筛选法从268株木霉菌中筛选获得两株对致病疫霉有较强抑菌活性的木霉菌株R-5和T-15。这两株木霉菌代谢液可抑制病原菌生长及孢子囊萌发。温室防病试验发现,接种两株木霉菌可以减轻晚疫病的发生。田间试验进一步证明,两株木霉菌对晚疫病具有良好的田间防治效果,防效分别达到了72.4%和70.0%。经分子生物学方法鉴定,两株木霉菌分别为拟康氏木霉和棘孢木霉。实验构建的以活体筛选为基础的生防木霉菌筛选方法是一种可行高效的生防木霉菌筛选方式。     

The production and characterisation of trichotoxin peptaibols, by Trichoderma asperellum

Trichoderma spp. are regularly found as a constituent of the mycoflora of many soils and are noted for their antagonistic activity against bacteria and other fungi. This latter property is the basis for the widespread interest in their use in the biological control of soil-borne fungal plant pathogens. This antagonism is partly based on their ability to produce an impressive inventory of secondary metabolites. An important group of bioactive metabolites produced by Trichoderma spp. are the non-ribosomal peptides (NRPs), especially the peptaibols. A virulent antagonistic strain, T. asperellum, which had been used in biological control strategies in Malaysia and previously examined for mycolytic enzyme production, has been studied for its potential for peptaibol production. The present research demonstrated the ability of T. asperellum to produce at least two metabolites which were identified as acid trichotoxin 1704E (Ac-Aib-Gly-Aib-Leu-Aib-Gln-Aib-Aib-Aib-Ala-Ala-Aib-Pro-Leu-Aib-Iva-Glu-Vol) and neutral trichotoxin 1717A (Ac-Aib-Gly-Aib-Leu-Aib-Gln-Aib-Aib-Aib-Ala-Aib-Aib-Pro-Leu-Aib-Iva-Gln-Vol). Addition of free Aib to the culture medium enhanced the production of trichotoxins. Biological activity of these substances was investigated against Bacillus stearothermophilus. The general characteristics of peptaibols, also found in the trichotoxins, include the presence of high proportions of the uncommon amino acid Aib, the protection of the N- and C-termini by an acetyl group and reduction of the C-terminus to 2-amino alcohols, respectively, amphipathy and microheterogeneity.     

棘孢木霉组合应用对杨树生长和光合特性影响

为研制复方棘孢木霉菌肥制剂,在大田条件下将等浓度（5×10³cfu·cm^-3土）不同组合的棘孢木霉（Trichoderma asperellum）分生孢子T1（Ta536+Ta4）、T2（Ta536+Ta4+Ta492）和T3（Ta536+Ta4+Ta492+Ta650）根施山新杨（Populus davidiana×P.alba var. Pyramidalis）组培移栽苗,研究棘孢木霉组合施用对山新杨生长及光合特性的影响。方差分析结果表明,诱导时间和不同处理对苗高、地径、叶绿素含量、叶绿素a/b值、P_n、Cond、C_i和T_r均有显著影响（P<0.05）：处理组的山新杨苗高、地径及干物质量均在不同程度上高于对照组（CK）,作用效果为T3 >T2 >T1 >CK;60d时,T3、T2和T1组杨树苗生物量分别比CK增加17.99%、14.28%和10.54%;15d时叶绿素含量比CK分别提高7.79%,6.91%和4.17%;叶绿素a/b值分别比CK提高7.79%、5.84%和4.73%。此外,处理组净光合速率（P_n）、胞间CO₂浓度（C_i）、气孔导度（Cond）、蒸腾速率（T_r）均高于CK,并且处理组的最大净光合速率、表观量子效率以及光饱和点均高于CK。同时,诱导时间和不同处理的交互作用对检测的多数指标（除P_n和C_i外）均具有显著影响（P<0.05）。说明4个棘孢木霉菌株组合施用具有协同作用,能提高菌株对环境条件的适应性,增强促生长和改善光合的作用效果。并且T3组对山新杨苗的影响最大、作用速度最快。     

Suppression of sugar beet damping-off caused by Rhizoctonia solani using bacterial and fungal antagonists

Rhizoctonia damping-off caused by Rhizoctonia solani Kühn, is one of the most damaging sugar beet diseases. It causes serious economic damage wherever sugar beets are grown. Biological control is an efficient and environmentally friendly way to prevent damping-off disease. Suppression of damping-off disease caused by R. solani was carried out by four isolates of Bacillus subtilis (Ehrenberg) Cohn as well as three isolates of each of Trichoderma harzianum Rifai and Trichoderma hamatum (Bonord.) Bainier. The effect of Bacillus and Trichoderma isolates against R. solani was investigated in vitro and tested on sugar beet plants under greenhouse conditions. Isolates of Bacillus and Trichoderma were able to inhibit the growth of R. solani in dual culture. Furthermore, Trichoderma isolates gave high antagonistic effect than isolates of B. subtilis. Under greenhouse conditions, coating seeds by T. harzianum and B. subtilis separately, reduced seedling damping-off significantly. However, applications of T. harzianum increased the percentage of surviving plants more than B. subtilis in comparison to control. The obtained results indicate that T. harzianum and B. subtilis are very effective biocontrol agents that offer potential benefit in sugar beet damping-off and should be harnessed for further biocontrol applications.     

棘孢木霉挥发性次级代谢产物检测及抑菌活性分析

木霉是一类具有重要生防价值的丝状真菌。文中首先对分离自浙江省绍兴市和广东省佛山市共12株棘孢木霉Trichoderma asperellum进行平板拮抗评价,然后采用顶空固相微萃取气质联用法(HS-SPME-GC-MS)检测拮抗性较好的两株菌的挥发性次级代谢产物。结果表明,棘孢木霉ZJSX5003和GDFS1009菌丝生长迅速,对尖孢镰孢菌Fusariumoxysporum抑制率分别达73%和74%。挥发性次级代谢产物主要是醇类和酮类,其中包含异丁醇、异戊醇、3-甲基-3-丁烯-1-醇、3-羟基-2-丁酮、2,3-丁二醇和6-正戊基-2H-吡喃-2-酮(6-PAP)。进一步通过体外抑菌试验,证实6-PAP具有较好的抑制尖孢镰孢菌的效果,为开发以木霉菌代谢产物如6-PAP为主要成分的生防制剂提供指导。     

Use of Trichoderma harzianum cultured on ground mesocarp fibre of oil-palm as seed treatment to control Pythium aphanidermatum,a causal agent of damping-off of Chinese kale seedling

Trichoderma harzianum, isolate T 01-22, was cultured on either sorghum grains, ground mesocarp fibre of oil-palm or oil-palm shell, both amended with urea fertilizer (100:1, w/w). T. harzianum cultured on ground mesocarp fibre was then used to coat seeds of Chinese kale (Brassica alboglabra Bailey) to control damping-off of seedlings caused by Pythium aphanidermatum. Biomass of T. harzianum cultured on ground mesocarp fibre of oil-palm was more effective than Captan and Benomyl, but less effective than Metalaxyl, in controlling damping-off of Chinese kale seedlings. Viability of T. harzianum growing on sorghum grains was reduced significantly during 7 months storage, followed by that of T. harzianum cultured on ground mesocarp fibre and oil-palm shell, both amended with urea fertilizer (46-0-0) at 100:1 (w/w).     

Biocontrol of Rhizoctonia solani and Pythium ultimum on Capsicum by Trichoderma koningii in potting medium.

Two isolates of Trichoderma koningii were evaluated for efficacy in control of damping-off diseases in seedlings of Capsicum annuum grown in pasteurized potting medium in a glasshouse. A selected isolate of binucleate Rhizoctonia and two fungicides were also included as standards for control of Rhizoctonia solani and Pythium ultimum var. sporangiiferum. Both isolates of T. koningii reduced seedling death caused by R. solani in one of two experiments, and by P. u. sporangii-ferum in two of three experiments. Neither isolate of T. koningii suppressed damping-off caused by either pathogen as consistently as the binucleate Rhizoctonia or fungicides. The implications of these results for commercial disease management are discussed.     

Diversity and antagonistic potential of Bacillus spp. associated to the rhizosphere of tomato for the management of Rhizoctonia solani

Bacillus spp. has emerged as the most effective alternative to synthetic chemical fungicides. To get a better insight in the antagonistic potential of Bacillus strains, rhizospheric soil samples of healthy tomato plants from Indo-gangetic plain regions of India were analysed. A total of 108 Bacillus strains were obtained from preliminary screening. Potent strains identified on the basis of in vitro antagonistic and biochemical assays were subjected to diversity analysis using 16S-rDNA, BOX and ERIC-PCR. Furthermore, the four best performing antagonistic Bacillus strains under in vitro plant growth promotion and antagonistic assay were selected for pot experiment. In field study, Bacillus amyloliquefaciens MB101 and Bacillus subtilis MB14 showed drastic reduction in disease index by 55.7 and 41.74% with significant elevation in fruit yield up to 220 and 184 qha^–1, respectively. The present study was successful in selecting effective Bacillus strains by performing phenotypic and genotypic characterisation of Bacillus strains that can be used as an integral component of integrated disease management of tomato root rot and damping-off.     

An immunological approach to quantifying the saprotrophic growth dynamics of Trichoderma species during antagonistic interactions with Rhizoctonia solani in a soil-less mix

Studies of the saprotrophic growth dynamics of Trichoderma species and their fungal hosts during antagonistic interactions are severely hampered by the absence of methods that allow the unambiguous identification and quantification of individual genera in complex environments such as soil or compost containing mixed populations of fungi. Furthermore, methods are required that allow discrimination between active hyphal growth and other components of fungal biomass such as quiescent spores that are produced in large numbers by Trichoderma species. This study details the use of monoclonal antibodies to quantify the saprotrophic growth dynamics of the soil-borne plant pathogen Rhizoctonia solani and biological control strains of Trichoderma asperellum and Trichoderma harzianum during antagonistic interactions in peat-based microcosms. Quantification was based on the immunological detection of constitutive, extracellular antigens that are secreted from the growing tip of Rhizoctonia and Trichoderma mycelium and, in the case of Trichoderma harzianum, from quiescent phialoconidia also. The Trichoderma-specific monoclonal antibody (MF2) binds to a protein epitope of the enzyme glucoamylase, which was shown by immunofluorescence and immunogold electron gold microscopy studies of Trichoderma virens in vitro to be produced at the origin of germ tube emergence in phialoconidia and from the growing tip of germ tubes. In addition, a non-destructive immunoblotting technique showed that the enzyme was secreted during active growth of Trichoderma asperellum mycelium in peat. The Rhizoctonia solani-specific monoclonal antibody (EH2) similarly binds to a protein epitope of a glycoprotein that is secreted during active mycelial growth. Extracts derived from lyophilized mycelium were used as a quantifiable and repeatable source of antigens for construction of calibration curves. These curves were used to convert the absorbance values obtained in ELISA tests of peat extracts to biomass equivalents, which allowed comparisons of the saprotrophic growth dynamics of the pathogen and antagonists to be made in single or mixed species microcosms. Trichoderma species were able to compete successfully with R. solani for nutrients and to prevent saprotrophic growth of the pathogen. Specificity of the Trichoderma quantitative assay was tested in non-sterile soil-based microcosms artificially inoculated with T. asperellum. The assay was highly specific and only detected T. asperellum population dynamics. No cross-reactivity was found with extracts from soil samples containing contaminant fungi.     

Enhanced activity of introduced biocontrol agents in solarized soils and its implications on the integrated control of tomato damping-off caused by Pythium spp

Soil solarization in combination with introduction of biocontrol agents (BCA) was evaluated as a potential disease management strategy for tomato damping-off caused by Pythium spp. A rifampicin resistant Pseudomonas fluorescens strain (PfT-8) and a carbendazim resistant Trichoderma harzianum strain (ThM-1) were introduced into soil following solarization. Tomato seeds were planted into treated field plots. The influence of soil solarization and application of biocontrol agents on damping-off incidence, plant biomass, rhizosphere population of introduced antagonists, and native Pythium spp. was assessed by two consecutive field trials. Damping-off incidence was significantly reduced in solarized plots compared to control. Soil inoculation of biocontrol agents into solarized plots resulted in the highest suppression of damping-off incidence (PfT-8 up to 92%; ThM-1 up to 83%), and increase in plant biomass (PfT-8 up to 66%; ThM-1 up to 48%) when compared to un-solarized control plots. Rhizosphere population of introduced biocontrol agents gradually increased (PfT-8 up to 102% and ThM-1 up to 84%) in solarized soils when compared to unsolarized control. The population of Pythium spp in rhizosphere soil was reduced up to 55% in solarized plots; whereas, application of BCA to solarized soils reduced the rhizosphere population of Pythium spp. by 86 and 82% in P. fluorescens and T. harzianum applied plots respectively.     

农杆菌介导生防棘孢木霉菌转化体系的优化

目的:实现棘孢木霉菌T4的遗传转化并优化其转化体系.方法:以潮霉素抗性为选择标记,利用农杆菌转化法介导转化棘孢木霉菌.结果:潮霉素基因成功整合到受体菌基因组中,转化子抗性基因可稳定遗传.结论:最优的转化体系和条件为:IM和CM培养基中AS浓度为200 μg/mL,棘孢木霉T4孢子浓度为106/mL,农杆菌浓度为200 μL( OD600约0.8),共培养时间为48 h,转化效率约为50个转化子/106个孢子.     

木霉属Trichoderma组和Pachybasium组的分子系统学研究

对来源不同的木霉及其有性型Longibrachiatum组、Trichoderma 组和Pachybasium组的81个菌株进行了ITS序列测定,并对ITS1序列用PHYLIP程序包中的DNAPARS程序进行系统发育分析。结果表明Trichoderma 组和Pachybasium组的所有菌株可分成两个群(A,B),B群进一步分为4个分支(B1,B2,B3,B4);A群由Trichoderma 组的H. aureoviridis和未鉴定到种的3个Hypocrea菌株构成;B1,B2,B4群均由Pachybasium组菌株构成;B3群由Pachybasium组的T. hamatum、T. strigosum和Trichoderma 组的T. asperellum、T. atroviride、T. koningii、T. viride和Hypocrea菌株T261构成。2个组相互交叉,组间没有明确的区分,进一步证明Pachybasium组是多系的。建议将Trichoderma 组中的T. viride aggr.、T. atroviride、T. koningii归并入Pachybasium组,对Trichoderma 组重新定义。