Identification of Trichoderma strains from building materials by ITS1 ribotyping, UP-PCR fingerprinting and UP-PCR cross hybridization

To study the role of Trichoderma in sick building syndrome, it is essential to be able to accurately identify species. Forty-four strains of Trichoderma spp. isolated from Danish buildings damaged by water leaks were identified using ITS1 ribotyping and universally primed PCR, UP-PCR. Ribotyping allowed the assignment of the strains into three distinct groups. High similarity of UP-PCR banding profiles of the strains allowed species designation for almost all strains (43 out of 44) when compared with the UP-PCR banding profiles obtained from reference strains of T. atroviride, T. citrinoviride, T. harzianum, T. longibrachiatum and T. viride. However, cross hybridization of UP-PCR products showed that the latter strain had high DNA homology to the ex-type strain of T. hamatum. The combined approach is a convenient way for reliable identification of Trichoderma strains.     

Strain-specific SCAR markers for the detection of Trichoderma harzianum AS12-2, a biological control agent against Rhizoctonia solani, the causal agent of rice sheath blight

In order to identify a specific marker for T. harzianum AS12-2, a strain capable of controlling rice sheath blight caused by Rhizoctonia solani, UP-PCR was performed using five universal primers (UP) both separately and in pairwise combinations. The application of two UP primers resulted in the amplification of unique fragments from the genomic DNA of T. harzianum AS12-2, clearly distinguishing it from other Trichoderma strains. The unique fragments had no significant sequence homology with any other known sequence available in databases. Based on the sequences of the unique fragments, 14 oligonucleotide primers were designed. Two primer sets amplified a fragment of expected size from the DNA of strain T. harzianum AS12-2 but not from any other examined strains belonging to T. harzianum, to other Trichoderma species assayed, or to other common fungi present in paddy fields of Mazandaran province, Iran. In conclusion, SCAR (sequence characterized amplified regions) markers were successfully identified and rapid, reliable tools were provided for the detection of an effective biocontrol Trichoderma strain, which can facilitate studies of its population dynamics and establishment after release into the natural environment.     

Molecular characterization and identification of biocontrol isolates of Trichoderma spp

The most common biological control agents (BCAs) of the genus Trichoderma have been reported to be strains of Trichoderma virens, T. harzianum, and T. viride. Since Trichoderma BCAs use different mechanisms of biocontrol, it is very important to explore the synergistic effects expressed by different genotypes for their practical use in agriculture. Characterization of 16 biocontrol strains, previously identified as "Trichoderma harzianum" Rifai and one biocontrol strain recognized as T. viride, was carried out using several molecular techniques. A certain degree of polymorphism was detected in hybridizations using a probe of mitochondrial DNA. Sequencing of internal transcribed spacers 1 and 2 (ITS1 and ITS2) revealed three different ITS lengths and four different sequence types. Phylogenetic analysis based on ITS1 sequences, including type strains of different species, clustered the 17 biocontrol strains into four groups: T. harzianum-T. inhamatum complex, T. longibrachiatum, T. asperellum, and T. atroviride-T. koningii complex. ITS2 sequences were also useful for locating the biocontrol strains in T. atroviride within the complex T. atroviride-T. koningii. None of the biocontrol strains studied corresponded to biotypes Th2 or Th4 of T. harzianum, which cause mushroom green mold. Correlation between different genotypes and potential biocontrol activity was studied under dual culturing of 17 BCAs in the presence of the phytopathogenic fungi Phoma betae, Rosellinia necatrix, Botrytis cinerea, and Fusarium oxysporum f. sp. dianthi in three different media.     

Characterization of Trichoderma Isolates from Philippine Rice Fields by UP-PCR and rDNA-ITS1 Analysis: Identification of UP-PCR Markers

Forty-two isolates of Trichoderma from rice fields in four provinces in the Philippines were characterized using rDNA-ITS1 analysis and universally primed polymerase chain reaction (UP-PCR). Two groups were clearly distinguishable on the basis of length and restriction pattern of the internal transcribed spacer (ITS) region of the ribosomal DNA and UP-PCR banding profiles using UP primer, L45. The 40 isolates comprising the largest group were very similar with respect to their UP-PCR banding profiles and were assigned to Trichoderma harzianum Rifai following morphological identification of four of the isolates. The two isolates belonging to the second group were identified as Trichodermaviride Pers. ex. Gray on the basis of their morphology, rDNA-ITS1 analysis and distinct UP-PCR banding profiles. One of the T. harzianum isolates with good cellulolytic and competitive saprophytic abilities was analysed using single and pair-wise combinations of UP primers in order to distinguish it from the remaining 41 isolates. A suitable diagnostic marker was identified and this marker will be valuable for monitoring the isolate in field tests.     

A new cultivation independent approach to detect and monitor common Trichoderma species in soils

A set of primers was developed for the detection, identification and quantification of common Trichoderma species in soil samples. Based on a broad range master alignment primers were derived to amplify an approximate 540 bp fragment comprising the internal transcribed spacer region 1 (ITS 1), 5.8S rDNA and internal transcribed spacer region 2 (ITS 2) from all taxonomic Clades of the genus Trichoderma. The primer set was applied to test strains as well as community DNA isolated from arable and forest soil. For all tested isolates the corresponding internal transcribed spacer regions of Trichoderma spp. strains were amplified, but none of non-Trichoderma origin. PCR with community DNA from soil yielded products of the expected size. Analysis of a clone library established for an arable site showed that all amplified sequences originated exclusively from Trichoderma species mainly being representatives of the Clades Hamatum, Harzianum and Pachybasioides and comprising most of the species known for biocontrol ability. In a realtime PCR approach the primer set uTf/uTr also proved to be a suitable system to quantify DNA of Trichoderma spp. in soils.     

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。     

Genetic relatedness of Trichoderma sect. Pachybasium species based on molecular approaches

Molecular approaches including internal transcribed spacer (ITS) sequences of ribosomal DNA, universal primer polymerase chain reaction (UP-PCR) fingerprinting, and DNA-DNA hybridization were used to study the genetic relatedness of species within Trichoderma sect. Pachybasium. In the analysis of ITS and 5.8S sequences of ribosomal DNA, parsimony analysis demonstrated that forty-one strains were distributed into five main groups supported by high bootstrap values. The species of Trichoderma sect. Pachybasium were clustered into groups I, II, and IV, with the strains of Trichoderma fasculatum and Trichoderma strictipile forming a separate branch, an independent group V. Some species within each group showed nearly identical sequence differences (fewer than 1-3 bp). UP-PCR and DNA-DNA hybridization were further used to clarify the genetic relatedness of these species with highly similar ITS sequences. Highly similar or identical UP-PCR profiles and high values of DNA complementarity (>70%) were observed among some species, Trichoderma hamatum and Trichoderma pubescens; Trichoderma croceum, Trichoderma polysporum and Trichoderma album, Trichoderma crassum and Trichoderma flavofuscum; and Trichoderma strictipile and Trichoderma fasciculatum. Although every species can be differentiated morphologically, the species showed highly similar molecular characteristics in the above cases, indicating that they could be conspecific. However, in some cases (Trithoderma longipile, T. crassum and T. flavofuscum; Trichoderma fertile and Trichoderma minutisporum; Trichoderma tomentosum, Trichoderma inhamatum and Trichoderma harzianum) there were discriminative patterns of UP-PCR and (or) low levels (<50%) of DNA-DNA hybridization; even their ITS sequences were similar, suggesting a closely phylogenetic relationship.     

Colony morphology mutants of Trichoderma harzianum with increased beta-1,4-N-acetyl-glucosaminidase production

A total of 36 UV-induced mutants with altered colony morphology were isolated from strain Trichoderma harzianum T334, a potential biocontrol agent against plant pathogenic fungi with the ability to produce constitutively low levels of chitinases. The level of constitutive beta-1,4-N-acetyl-glucosaminidase production in standing and shaken cultures under non-inductive conditions was tested in mutants and compared to that of the parental strain. About 30% of the mutants showed significantly increased levels of enzyme production, with strain T334 col26a being the best producer. This mutant and the parental strain were subjected to in vitro confrontation assays with plant pathogenic Fusarium culmorum, Pythium debaryanum and Rhizoctonia solani strains. The mutant derivative could be characterized by significantly higher biocontrol index values than the parental strain in each experiment, suggesting, that mutants with improved constitutive extracellular chitinase secretion could be applied for biocontrol purposes against fungal plant pathogens.     

Control Alternatives for Damping-Off in Tomato Seedling Production

In two tomato genotypes, we assessed control alternatives for damping-off with combinations of chemical fungicides and native/commercial strains of biological agents. Forty treatments consisting of 19 levels of mixing products, chemical fungicides, native strains and commercial products from biological control agents, and untreated treatment were used onto Ramsés and Toro hybrids. They were distributed on an incomplete block design in divided plots arrangement, where genotypes constitute the larger ones and the 8-repetition mixed products, the smaller ones. Putting 180 mL of fungal complexes, made of spores and mycellium Fusarium-solani (2 × 106 UFC), Rhizoctonia-solani (1 × 106 UFC), Phytophthora-capsici (1 × 105 UFC) and Sclerotium-rolfsii (mycellium-sclerotia) on each seedling trays, made inoculation possible. The mixtures of (Bacillus spp. + Streptomyces spp. + Trichoderma spp.) + (Propamocarb + Fosetyl); (Bacillus spp. + Streptomyces spp. + Trichoderma spp.) + (Metalaxyl + Chlorothalonil); Pseudomonas fluorescens + Streptomyces + Micromonospore + Sporideamium + Aminoacidos, Péptidos, Carbohidratos) + (Propamocarb + Fosetyl); the native strain of Trichoderma asperellum + (Propamocarb + fosetil) and the native strains Trichoderma asperellum + Bacillus subtilis, diminished damping-off, prevented its appearance and had most significant agronomic characteristics. In contrast to this, combination of (Mancozeb) + (Free iodine) + (Metalaxyl + Chlorothalonil) + (Methyl thiophanate) produced some toxicity in the plant. In addition, Ramsés presented the best agronomic parameters, while Toro had the utmost fresh and dry weight in root.     

Biological and Integrated Control of Botrytis Bunch Rot of Grape UsingTrichodermaspp.

Field trials were carried out in upstate New York in 1990, 1992, 1993, and 1994 and in Chile in 1992–1993 and 1993–1994 in order to evaluate the ability of various strains ofTrichodermaspp. to control bunch rot of grape, to assess the compatibility and possible additive effects of selected biocontrol fungi and dicarboximide fungicides, and to determine factors affecting biocontrol efficacy. In 1990, three strains ofTrichodermaspp. were evaluated for their biocontrol ability, and all provided significant control ofBotrytis cinerea.As few as two late applications of the biocontrol fungi were nearly as effective as up to five applications throughout bloom and fruit development. Trials in New York in 1992 and in Chile in 1992–1993 indicated thatTrichoderma harzianumcould replace some applications of iprodione or vinclozolin with little reduction in efficacy. In New York in 1993, we found that applications ofT. harzianumat bloom and early fruit development followed by a tank-mix application ofT. harzianumand half rates of iprodione gave extremely effective control of bunch rot. In 1994, less effective control was obtained than in earlier years. Addition of a nutritive adhesive (Pelgel, a mixture of carboxymethyl cellulose and gum arabic) applied with the biocontrol agent tended to improve results. Thus, biological control of bunch rot of grape withT. harzianumcan be an effective method of management of this disease.     

Major secondary metabolites produced by two commercial Trichoderma strains active against different phytopathogens

AIMS: Trichoderma harzianum strains T22 and T39 are two micro-organisms used as active agents in a variety of commercial biopesticides and biofertilizers and widely applied amongst field and greenhouse crops. The production, isolation, biological and chemical characterization of the main secondary metabolites produced by these strains are investigated. METHODS AND RESULTS: Of the three major compounds produced by strain T22, one is a new azaphilone that shows marked in vitro inhibition of Rhizoctonia solani, Pythium ultimum and Gaeumannomyces graminis var. tritici. In turn, filtrates from strain T39 were demonstrated to contain two compounds previously isolated from other T. harzianum strains and a new butenolide. The production of the isolated metabolites was also monitored by liquid chromatography/mass spectrometry during in vitro interaction with R. solani. CONCLUSIONS: This paper reports the isolation and characterization of the main secondary metabolites obtained from culture filtrates of two T. harzianum strains and their production during antagonistic interaction with the pathogen R. solani. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first work on secondary metabolites produced by the commercially applied strains T22 and T39. Our results provide a better understanding of the metabolism of these fungi, which are both widely used as biopesticides and/or biofertilizers in biocontrol.     

The prevalence of campylobacters and arcobacters in broiler chickens

Chicken carcasses from a supermarket and from a poultry abattoir were examined using methods designed to isolate as many strains of campylobacters and related organisms as possible. Strains of arcobacter, but no campylobacters, were isolated from every carcass after enrichment. Campylobacter jejuni subsp. jejuni was isolated from all carcasses examined by direct plating and other Campylobacter -like strains were isolated from nine out of 15 abattoir carcasses by direct plating but not after enrichment. Only the Camp. jejuni subsp. jejuni strains could be identified to species level using a readily available identification scheme and/or a commercial identification kit. Examination of caecal contents from the 15 abattoir poultry yielded Camp. jejuni subsp. jejuni and Campylobacter -like strains from 15 and eight by direct plating, and from six and nine after enrichment, respectively. Four sites in the intestine of the abattoir birds (60 samples) were examined for arcobacters and only one strain was isolated. This indicates that arcobacters are probably not normal inhabitants of the poultry intestine. Poultry is a rich source of other campylobacteria besides the thermophilic Campylobacter spp.     

Nursery treatments with resistant inducers,soil amendments and biocontrol agents for the management of the Fusarium wilt of lettuce under glasshouse and field conditions

Lettuce Fusarium wilt, caused by Fusarium oxysporum f. sp. lactucae, represents a major problem in most lettuce production areas worldwide. In the present study, a number of resistance inducers, organic amendments and biocontrol agents were applied in a preventative way, in experimental and commercial situations, to soils artificially or naturally infested with race 1 of the pathogen, and to moderately susceptible lettuce cultivars. Potassium phosphite, acibenzolar‐S‐methyl, green composts, and Bacillus subtilis Qst713, Trichoderma asperellum + Trichoderma gamsii, and Pseudomonas strains achieved the most consistent disease control under the experimental conditions. Moreover, potassium phosphite, green compost, B. subtilis Qst713 and T. asperellum + T. gamsii, also showed a positive effect on plant development. In general, the results of the different treatments in naturally infested soil were similar to those observed in glasshouse trials under artificial inoculation. Potassium phosphite provided a consistent disease reduction (48%–62% in artificially infested soil and 60%–75% in naturally infested soil). The effects of adding 10% compost to a peat growing medium in the nursery, followed by a soil mixing application when lettuce was transplanted, significantly reduced the severity of Fusarium wilt (50%–59% efficacy) and increased fresh biomass production. Compost enrichment with Trichoderma TW2 generally further increased its efficacy. When tested under field conditions, the commercially available Trichoderma spp. and B. subtilis, together with experimental strains of Pseudomonas and Trichoderma spp., applied at the nursery level, provided a disease reduction of 30%–78%. Early application of the different control measures under nursery conditions and at lettuce transplant is noteworthy because it was carried out at a more localized level, with reduced amounts of products. Their use in practice should be integrated with other control strategies.     

洞庭湖湿地木霉多样性及生防活性

【目的】了解湖南省洞庭湖湿地木霉种类及分布,丰富我国的木霉种质资源,为功能菌株筛选应用奠定基础。【方法】利用ITS序列比对分析结合形态学特征对分离到的木霉菌株进行种类鉴定,构建系统发育进化树分析其亲缘关系。通过菌丝生长速率法测定菌株的平板抑菌能力,根据水解带大小检测菌株的水解酶活性,利用灰色关联度分析筛选综合生防效果较好的木霉菌株。【结果】从52个土样和18个水样中分离得到114株木霉菌株,经鉴定分属16个木霉种类:哈茨木霉、绿木霉、刺孢木霉、土星孢木霉、钩状木霉、拟康宁木霉、短密木霉、深绿木霉、猥木霉、毛细木霉(中国新记录种)、长枝木霉、卵孢木霉、侧耳木霉、加纳木霉、厚木霉及一个疑似新种;哈茨木霉为洞庭湖湿地中的优势种,占总菌株数量的19.30%;16种木霉在系统发育树中归于7个进化支:Harzianum Clade、Virens Clade、Longibrachiatum Clade、Lutea Clade、Viride Clade、Hamatum Clade、Unknown Clade。灰色关联度分析表明,菌株TW21990、QT22040和QT22094的灰色关联度较高,分别为0.849 5、0.798 6和0.732 6,综合生防性状较好。【结论】洞庭湖湿地木霉具有种类多样性和分布多样性,发现了一个中国新记录种毛细木霉和一个疑似新种,哈茨木霉TW21990、长枝木霉QT22040和卵孢木霉QT22094是潜在的优良生防菌株。     

拮抗木霉耐多菌灵菌株的筛选

该文进行了耐多菌灵生防木霉菌株的筛选和生防效果的测定研究。为获得对灰葡萄孢霉等病原菌有显著拮抗能力的耐多菌灵木霉菌株,通过含药平板诱导和紫外线诱变,获得5株耐药性菌株,在多菌灵2.0g/L浓度下生长较好,Ec50值提高了40多倍;抗性菌株在含药培养基上连续继代转移培养10次,抗性程度未见下降;经过平板对峙和活体拮抗性测定,确定T42-2为最佳拮抗性耐多菌灵菌株,其对黄瓜灰霉病的防效达92.13%,并且对多菌灵、速克灵和三唑铜存在交互抗性。     

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.     

Three native Pseudomonas fluorescens strains tested under growth chamber and field conditions as biocontrol agents against damping-off in alfalfa

Alfalfa (Medicago sativa) is one of the most important crops used in Uruguay for livestock feeding. Seedling diseases, particularly damping-off, are a critical factor which limits its establishment. Three native Pseudomonas fluorescens strains, UP61.2, UP143.8 and UP148.2, previously isolated from Lotus corniculatus, were evaluated to determine their efficacy as biological control agents for alfalfa seedling diseases in the field. Their compatibility with the alfalfa-Sinorhizobium meliloti symbiosis was also assessed. In growth chamber conditions seed inoculation with Pseudomonas strains did not affect different parameters of alfalfa-rhizobium symbiosis as shown by nodulation rate and shoot dry weight of plants. The presence of the commercial inoculant strains of S. meliloti did not impair colonization by the P. fluorescens and vice versa. In field trials the dynamics of rhizobial rhizospheric populations were not affected by the presence of P. fluorescens. Each P. fluorescens strain successfully colonized alfalfa roots at adequate densities for biocontrol activity. Results showed that P. fluorescens strains provided a 10–13% increase in the number of established plants relative to the control, an intermediate result compared to the fungicide treatment (24%). The alfalfa above-ground biomass was increased by 13% and 15–18% in the presence of the fungicide and P. fluorescens strains, respectively. Therefore, results from this study demonstrated that the three P. fluorescens strains provided effective control against soil-borne pathogens and suggest a potential use in the development of a commercial inoculant to be applied for the control of legume seedling diseases.     

一株具有拮抗作用的解淀粉芽孢杆菌的筛选、鉴定及生物学特性研究

本研究以樱桃番茄成熟红果为样本,以极细链格孢菌为指示菌筛选得到一株对番茄采后病原菌有抑制作用的菌株KL-1。通过平板拮抗试验研究了菌株KL-1对番茄采后常见病原真菌极细链格孢菌、黑曲霉、青霉菌、尖侧多隔孢霉、拟康宁木霉、盐生枝孢霉、子囊菌、胶孢炭疽菌等八种病原菌的抑制作用,同时通过形态学、生理生化及16S rDNA分子生物学特征对菌株进行了鉴定,并对其基本生物学特性进行了研究。结果表明:菌株KL-1对于七种病原菌极细链格孢菌、黑曲霉、青霉菌、拟康宁木霉、盐生枝孢霉、子囊菌和胶孢炭疽菌均有明显的抑制作用,其中对黑曲霉的抑制率最大为81%,对其他六种病原菌抑制率也均高于70%,对尖侧多隔孢霉的抑制率为0,说明菌株KL-1可以抑制番茄多种常见病原真菌,具有开发为生防制剂的潜力。经鉴定KL-1为解淀粉芽孢杆菌(Bacillus amyloliquefaciens);该菌培养9 h内生长最旺盛,最适生长pH为7.0,最适生长温度为37℃。     

Molecular Characterization and Identification of Biocontrol Isolates of Trichoderma spp.

The most common biological control agents (BCAs) of the genus Trichoderma have been reported to be strains of Trichoderma virens, T. harzianum, and T. viride. Since Trichoderma BCAs use different mechanisms of biocontrol, it is very important to explore the synergistic effects expressed by different genotypes for their practical use in agriculture. Characterization of 16 biocontrol strains, previously identified as “Trichoderma harzianum” Rifai and one biocontrol strain recognized as T. viride, was carried out using several molecular techniques. A certain degree of polymorphism was detected in hybridizations using a probe of mitochondrial DNA. Sequencing of internal transcribed spacers 1 and 2 (ITS1 and ITS2) revealed three different ITS lengths and four different sequence types. Phylogenetic analysis based on ITS1 sequences, including type strains of different species, clustered the 17 biocontrol strains into four groups: T. harzianum-T. inhamatum complex, T. longibrachiatum, T. asperellum, and T. atroviride-T. koningii complex. ITS2 sequences were also useful for locating the biocontrol strains in T. atroviride within the complex T. atroviride-T. koningii. None of the biocontrol strains studied corresponded to biotypes Th2 or Th4 of T. harzianum, which cause mushroom green mold. Correlation between different genotypes and potential biocontrol activity was studied under dual culturing of 17 BCAs in the presence of the phytopathogenic fungi Phoma betae, Rosellinia necatrix, Botrytis cinerea, and Fusarium oxysporum f. sp. dianthi in three different media.