Use of Honey Bees and Bumble Bees to Disseminate Trichoderma harzianum 1295-22 to Strawberries for Botrytis Control

The effectiveness of using honey bees and bumble bees to vector a commercial formulation of Trichoderma harzianum 1295-22 for the control of Botrytis cinerea on strawberries was evaluated from 1994 to 1997 in 2 strawberry fields at the New York State Agricultural Experiment Station in Geneva, New York and in 10 grower fields in eight counties of New York. Commercial bumble bee colonies were used to deliver the biocontrol agent in 1994 and 1995 and five-frame nuclear honey bee hives were used in 1995–1997. Each honey bee exiting the hive carried about 1 × 10⁵ colony-forming units of T. harzianum, with the majority found on the bees' legs (58%). Flowers collected from the bee-delivered treatment generally had half the density of T. harzianum as those from the sprayed treatment. However, during the 4 years of this study, T. harzianum delivered by bumble bees or honey bees provided better Botrytis control than that applied as a spray. In addition, the bee-delivered T. harzianum provided the same or a better level of control of Botrytis as commercial fungicides applied at bloom. Strawberries collected from the bee-visited treatments averaged 22% more seeds and weighed between 26 and 40% more than berries in nonvisited treatments. The number of seeds per berry and berry weight were reduced by 7–12% in plots treated with fungicides and visited by bees, indicating that the use of some commercial fungicides at bloom may impact pollination and yield. Bee delivery of T. harzianum 1295-22 is a viable option for strawberry growers interested in controlling Botrytis with minimal fungicide use.     

Effect of<Emphasis Type="Italic">Trichoderma</Emphasis> species on growth of Fusarium<Emphasis Type="Italic">proliferatiom</Emphasis> and production of fumonisins,fusaproliferin and beauvericin

Trichoderma species has been suggested as potential biocontrol agent forFusarium verticillioides on maize. In this cereal,F. verticillioides and F. proliferatum contributed to fumonisin accumulation. In addition,F. proliferatum could produce beauvericin and fusaproliferin. The aim of this work was to evaluate the effect ofTrichoderma spp. on growth and fumonisin B¹ fusaproliferin and beauvericin production byF. proliferatum. Dual cultures of F.proliferatum andT. harzianum ITEM 3636 andT. longibrachiatum ITEM 3635 on maize meal agar at 0.995 aw were done. The effect ofTrichoderma spp. on the lineal growth ofF. proliferatum was determined. The effect ofTrichoderma species on fumonisin B¹, fusaproliferin and beauvericin production byF. proliferatum was determined on co-inoculated maize kernels by HPLC.T. harzianum suppressedF. proliferatum growth once contact between the colonies occurred.T. longibrachiatum showed a less antagonistic effect againstF. proliferatum. A reduction on fumonisin B¹ production of 98% and 88% was observed in the co-incubation ofF. proliferatum withT. harzianum andT. longibrachiatum, respectively. The decrease of FB¹ production was significant even in maize kernels on whichF. proliferatum had been growing 7 days prior to the addition ofTrichoderma spp. The concentration of beauvericin and fusaproliferin produced during 30 days coincubation ofF. proliferatum with bothTrichoderma spp. did not differ to those produced byF. proliferatum alone. These mycotoxins might enter the food chain causing so far unknown consequences to the health of domestic animals and humans. For this reason it is important, when a potential biocontrol agent is under study, to test the effect on the fungal growth and on the putative mycotoxin produced. Part of the information was presented at the Mycotoxin Prevention Cluster Dissemination Day and Mycoglobe Launch Conference, Brussels, Belgium, Oct 20–21, 2004 Financial support: Agenda Córdoba Ciencia, grant N^o 0279–000431/00     

Control of the bean rust fungus <Emphasis Type="Italic">Uromyces appendiculatus</Emphasis> by means of <Emphasis Type="Italic">Trichoderma harzianum</Emphasis>: leaf disc assays on the antibiotic effect of spore suspensions and culture filtrates

Several species of the fungal genus Trichoderma act as antagonists of other fungi. A number of strains from the Trichoderma species T. harzianum Rifai are used as biological control agents for the control of soilborne as well as foliar plant pathogens. Six T. harzianum strains, five of them isolated from commercial preparations, were evaluated for their capability to control the bean rust fungus Uromyces appendiculatus (Pers. ex Pers.) Unger. Different kinds of leaf disc assays were performed with conidial spore suspensions and sterile culture filtrates of the T. harzianum strains. Great differences were observed concerning the efficacy of the Trichoderma strains to reduce the number of the uredial pustules developing after rust inoculation which followed the application of the particular Trichoderma strains. Efficacy values ranged from 1 to over 50%. Increasing spore or culture filtrate concentrations of the two most effective isolates T12 and T_U led to decreases in the number of developing uredial pustules. Culture filtrate applications had a protective but no curative effect. T12 spore suspensions maintained their disease reducing activity even when autoclaved. This and some other evidence for an antibiotic interaction between T. harzianum and U. appendiculatus are discussed. Handling Editor: Reijo Karjalainen.     

Effect of peat-bran inoculum ofTrichoderma species on biological control ofRhizoctonia solani in lettuce

A range of known biocontrol or plant growth-stimulating species ofTrichoderma orGliocladium were grown on peat-bran substrate to yield between 5×10⁷–3×10¹⁰ colony forming units (cfu's)g^–1 substrate after 14 days growth. Inocula were incorporated into peat:sand potting compost infested withRhizoctonia solani to give 7–8 × 10⁴ cfu's of antagonist g^–1 compost and assessed for biological control activity using lettuce seedlings. Six of the eight antagonists decreased daming-off and three of these consistently increased yield in comparison withR. solani treatment alone.Subsequently, peat-bran inoculum ofT. harzianum isolate TH1 was incorporated at 0.5% w/v intoR. solani infested potting compost. Both autoclaved and nonautoclaved inoculum ofT. harzianum TH1 decreased disease and increased yield. Incorporation of ethyl acetate-extracted autoclaved inoculum ofT. harzianum TH1 resulted in similar levels of biocontrol and improved plant growth as did incorporation of nonautoclaved and autoclavedT. harzianum TH1 inoculum. The need to standardize inocula and controls is emphasized.     

Biocontrol of onion white rot by application of Trichoderma species formulated on wheat bran powder

In vitro, Trichoderma album, Trichoderma harzianum, Trichoderma koningii, Trichoderma viride and Trichoderma virens showed antagonistic effect against the most pathogenic isolate (Sc2) of Sclerotium cepivorum, the cause of onion white rot disease. Five Trichoderma preparations of each Trichoderma sp. were prepared on wheat bran powder to be used for controlling white rot disease of onion. Greenhouse and field experiments followed the same trend where T. harzianum and T. koningii were the most effective in reducing the incidence and severity of white rot disease compared with the control. Trichoderma species preparations caused promotion to vegetative parameters of onion plants in pots and increase bulb productivity in filed. In this regard, T. harzianum and T. koningii were the most effective. A positive correlation was found between the biocontrol activity of Trichoderma species preparations and enhancement of peroxidase, polyphenoloxidase and chitinase enzymes in onion plants to resist infection with S. cepivorum.     

Potential of genes and gene products fromTrichoderma sp. andGliocladium sp. for the development of biological pesticides

Fungal cell wall degrading enzymes produced by the biocontrol fungiTrichoderma harzianum andGliocladium virens are strong inhibitors of spore germination and hyphal elongation of a number of phytopathogenic fungi. The purified enzymes include chitinolytic enzymes with different modes of action or different substrate specificity and glucanolytic enzymes with exo-activity. A variety of synergistic interactions were found when different enzymes were combined or associated with biotic or abiotic antifungal agents. The levels of inhibition obtained by using enzyme combinations were, in some cases, comparable with commercial fungicides. Moreover, the antifungal interaction between enzymes and common fungicides allowed the reduction of the chemical doses up to 200-fold. Chitinolytic and glucanolytic enzymes fromT. harzianum were able to improve substantially the antifungal ability of a biocontrol strain ofEnterobacter cloacae. DNA fragments containing genes encoding for different chitinolytic enzymes were isolated from a cDNA library ofT. harzianum and cloned for mechanistic studies and biocontrol purposes. Our results provide additional information on the role of lytic enzymes in processes of biocontrol and strongly suggest the use of lytic enzymes and their genes for biological control of plant diseases.     

Assessment of the biocontrol potential of a Trichoderma viride isolate: Part II: Protection against soft rot and basidiomycete decay

A previous paper reported on the establishment of a field and fungal cellar trial set up to determine the biocontrol potential of a specific Trichoderma isolate against wood decay fungi. This paper reports on the analyses used to examine the protective effect of the selected isolate, and presents results indicating an initial protective effect against both basidiomycetes and soft rot fungi. The parameters assessed in the field and fungal cellar trials were soft rot decay, basidiomycete decay, Trichoderma colonisation, moisture content and nitrogen content. The results of these analyses show that the introduction of a biological control agent has had a significant effect on moisture content, decay and nitrogen content. A protective effect has been observed against soft rot and basidiomycete decay fungi in field samples.     

Biological efficacy of <Emphasis Type="Italic">Trichoderma harzianum</Emphasis> isolate to control some fungal pathogens of wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis>) in Turkey

Gaeumannomyces graminis var. tritici, Fusarium culmorum and F. moniliforme are highly important and widespread pathogens of wheat in Turkey. Trichoderma isolates have been used as biocontrol agents to protect plants against soilborne diseases in several crops. The present work was carried out to evaluate the potential of Trichoderma harzianum isolate T1 as biocontrol agents for G. graminis, F. culmorum and F. moniliforme under field conditions in 2001 and 2002. Quantitative differences were found in microbial number in soil. T. harzianum T1 had considerable effect on population densities of the tested pathogens. The total number of G. graminis, F. culmorum and F. moniliforme were lower in the T. harzianum T1 application made to seed. T. harzianum T1 application to seed had increasing affect on the yield components of wheat through better control over pathogens. The greatest counts of T. harzianum T1 were detected on root segments. Seed application by T. harzianum T1 had increasing effect on yield components of wheat.     

拮抗北细辛菌核病木霉菌的分离、鉴定及生防效果

【背景】菌核病是北细辛根部主要病害之一,木霉菌作为目前应用最广泛的生物防治真菌,利用木霉菌防治北细辛菌核病是目前研究的热点。【目的】通过稀释分离法对健康北细辛植株根际土壤进行菌株分离,以期筛选出有效拮抗北细辛菌核病的生防木霉菌。【方法】以北细辛菌核病菌为靶标菌,采用平板对峙培养、挥发性与非挥发性物质抑菌的方法对分离得到的木霉菌进行筛选,采用生长速率法对筛选出的木霉菌的发酵液进行抑菌效果测定,并采用硫代巴比妥酸法测定筛选出的木霉对北细辛菌核病菌的丙二醛(Malondialdehyde,MDA)含量、紫外吸收法测定过氧化氢酶(Catalase,CAT)活性、氮蓝四唑法测定超氧化物歧化酶(Superoxide Dismutase,SOD)活性、愈创木酚法测定过氧化物酶(Peroxidase,POD)活性的影响。【结果】从土壤中分离出木霉菌共14株,通过形态学和ITS-RPB2双基因联合构建系统发育树,鉴定其为哈茨木霉(Trichoderma harzianum)、钩状木霉(Trichoderma hamatum)、拟康氏木霉(Trichoderma koningiopsis)、深绿木霉(Tr...     

Effect of <Emphasis Type="Italic">Trichoderma</Emphasis> spp. isolates for biological control of tan spot of wheat caused by <Emphasis Type="Italic">Pyrenophora tritici-repentis</Emphasis> under field conditions in Argentina

Trichoderma spp. have been used as biocontrol agents to protect plants against foliar diseases in several crops, but information from field assays is scarce. In the present work, experiments were carried out to determine the effect of six isolates of Trichoderma harzianum and one isolate of T. koningii on the incidence and severity of tan spot, caused by Pyrenophora tritici-repentis (anamorph: Drechslera tritici-repentis) under field conditions. Significant differences between years, wheat cultivars and treatments were found. In 2003, two of the isolates assayed (T5, T7) showed the best performance against the disease applied as seed treatments or sprayed onto wheat leaves at different stages. The application of six of the treatments on wheat plants significantly reduced disease severity by 16 to 35% in comparison with the control. Disease control provided by isolate T7 was similar to that provided by the fungicide treatment (56% reduction). This is the first report on the efficacy of Trichoderma spp. against tan spot under field conditions in Argentina.     

Geographic range, impact, and parasitism of lepidopteran species associated with the invasive weed Lantana camara in South Africa

Six isolates of Trichoderma were screened for antagonism to Armillaria in tea stem sections buried in the soil. The inability of Armillaria to invade Trichoderma-colonized stem sections and the reduction of its viability in the plant materials following invasion of these by Trichoderma were used as indicators of antagonism. Four isolates of the species Trichoderma harzianum significantly (P<0.001) reduced the incidence of the pathogen in the plant materials. Isolate T4 completely eliminated the pathogen from plant materials in sterile soil and also antagonized two different isolates of the pathogen in nonsterile soil. Application of this T. harzianum isolate to the soil as a wheat bran culture significantly (P<0.001) reduced viability of Armillaria in woody blocks of inoculum. Soil amendment with coffee pulp also reduced the inoculum viability but did not affect the incidence of Trichoderma in the blocks of inoculum. We conclude that the direct application of wheat bran-formulated T. harzianum into soil surrounding woody Armillaria inoculum sources can suppress the pathogen. Further, no organic amendment is needed to enhance development of the antagonist in the soil as a pre-requisite to suppressing the pathogen.     

Development of Pusa 5SD for seed dressing and Pusa Biopellet 10G for soil application formulations of Trichoderma harzianum and their evaluation for integrated management of dry root rot of mungbean (Vigna radiata)

Various seed dressing and soil application formulations were developed from Trichoderma viride, T. virens and T. harzianum to increase the shelf life of bio-formulations used to manage dry root rot (Rhizoctonia bataticola) of mungbean (Vigna radiata), a major yield limiting factor in mungbean production. The shelf life of the formulations developed in the present study was monitored by counting colony forming units (cfu) up to 25 months of storage at room temperature (26 ± 8 °C). A newly developed seed dressing formulation, Pusa 5SD based on peat powder (47.5%), Sabudana powder (Manihot esculenta) (47.5%) and carboxymethyl cellulose (5%) and a newly developed soil application formulation, Pusa Biopellet (PBP) based on sodium alginate, aluminium silicate, Sabudana powder and tap water (1:5:5:100 w/w/w/v) exhibited longer shelf life. Another formulation Pusa Biogranule (PBG) based on wheat and pulse brans varied in cfu counts during different periods of storage. Pusa 5SD could be used up to 25 months of storage while PBP 10G and PBG 5 could be used up to 15 months of storage (>10⁵ cfu). The efficacy of the formulations was evaluated in pot experiments against the disease. In these experiments, T. harzianum based PBP 10G and PBG 5 for soil application, and Pusa 5SD for seed treatment were found to be superior to others in reducing the dry root rot incidence, and increasing the seed germination and shoot and root lengths. However, a combination of soil application of PBP 10G (T. harzianum) and seed treatment with T. harzianum based Pusa 5SD + carboxin was found superior to the use of any of these formulations alone in reducing the dry root rot incidence (87.2%) and increasing the seed germination (43.0%), shoot length (40.3%), root length (37.0%) and grain yield (54.6%) of mungbean crop over those of untreated control under sick field conditions.     

Establishment ofNeltumius arizonensis(Coleoptera: Bruchidae) on Mesquite (ProsopisSpecies: Mimosaceae) in South Africa

Species ofProsopis(Mimosaceae), or mesquites, are invasive rangeland weeds in South Africa's Western Cape and Northern Cape Provinces. Two bruchid seed-weevil species,Algarobius prosopis(Le Conte) andA. bottimeriKingsolver, were released for biological control in 1987 and 1990, respectively. Seed-feeding biocontrol agents were selected because mesquite pods are valued as livestock fodder. Livestock grazing of bruchid larvae developing in mesquite seeds, however, limits the effectiveness of these agents. Livestock grazing also exacerbates mesquite infestations because scarified seeds are dispersed widely in vertebrate dung. In response to the livestock grazing problem,Neltumius arizonensis(Schaeffer), a bruchid reputed to be capable of ovipositing on immature, tree-borne pods, was released at three sites in Western Cape Province in 1993 and 1994. Small populations ofN. arizonensishave become established at the release sites. Overall,N. arizonensiswas 18 times less abundant thanA. prosopis.In some monthsN. arizonensiseggs were heavily parasitized byUscanasp. (Trichogrammatidae), but the effect of this onN. arizonensispopulation dynamics is uncertain. Western CapeN. arizonensispopulations need more time to increase in size. The introduction of other, more injurious biocontrol agents such as the cecidomyiid bud feederAsphondylia prosopidisCockerell should be considered.     

The growth form of the inducing microorganism and chitin addition affect mycolytic enzyme production byTrichoderma harzianum

The effect of the growth form of the inducing microorganism on specificTrichoderma harzianum mycolytic enzyme production was studied. The pelleted form ofRhizopus nigricans gave a better product concerning protoplast formation ability. The maximum yield of protoplasts from the target fungusCochliobolus lunatus was 1×10⁸ ml^–1. Analysis of individual specific enzyme activities inTrichoderma mycolytic enzyme preparations confirms the importance of high chitinase and low protease activity for high protoplast yields. Supplementation of the production medium with chitin increased the chitinase activity in theTrichoderma exoenzyme mixture.     

关防风根腐病拮抗细菌筛选与鉴定

【背景】由木贼镰刀菌[Fusarium equiseti(Corda)Sacc.]引起的关防风根腐病,是近年来导致关防风产量及质量下降的主要土传病害之一。生物防治因其对环境安全及人畜无害等优势,成为目前植物病害防治的一种有效手段。【目的】挖掘关防风根际土壤中对木贼镰刀菌具有良好拮抗作用的生防菌株。【方法】采用稀释平板法分离根际土壤细菌;用滤纸片法和牛津杯法对拮抗细菌进行筛选和抑菌谱检测;用抗生素标记法标记拮抗细菌并测定其定殖能力;通过盆栽实验研究其对关防风根腐病的防效;通过形态学、生理生化特征及16S rRNA基因序列确定分类学地位。【结果】从健康关防风根际土壤中分离纯化了157株细菌,筛选获得对关防风根腐病菌具有显著抑菌作用的拮抗细菌SC-119,无菌滤液的抑菌率可达68.53%,而且兼具广谱抑菌能力和良好的定殖能力;盆栽实验表明,菌株SC-119对关防风根腐病的防治效果达到67.39%,其相对防效较接种哈茨木霉菌剂、枯草芽胞杆菌菌剂和代森锰锌分别提高了29.03%、32.26%和16.13%;对菌株SC-119进行分类学鉴定,确定其为萎缩芽胞杆菌(Bacillus atrophae...     

Foliar Application of Fungal Biocontrol Agents for the Control of White Mold of Dry Bean Caused by Sclerotinia sclerotiorum

Field experiments were conducted during 1992–1994 to evaluate the effectiveness of five indigenous fungi for control of white mold (Sclerotinia sclerotiorum) of dry bean (Phaseolus vulgaris). The five fungi consisted of one antagonist, Epicoccum purpurascens, and four mycoparasites, Coniothyrium minitans, Talaromyces flavus, Trichothecium roseum, and Trichoderma virens. Spore suspensions of each fungus were sprayed onto bean plants two or three times during the early bloom to midbloom period. Incidence of white mold of dry bean was significantly reduced by all biocontrol agents. C. minitans and E. purpurascens, the most effective agents, reduced the proportion of plants infected by an average of 56 and 43%, respectively (P < 0.001). C. minitans was the only biocontrol agent recovered consistently from sclerotia and diseased seed present in harvested samples. It was recovered at similar frequencies in samples from all treatments. Of the sclerotia of S. sclerotiorum collected from harvested seed, 59% were infected by C. minitans in 1993 and 20% were infected by C. minitans in 1994. In three additional trials in 1994, comparing C. minitans with the fungicide benomyl, the fungus was not effective in any of the experiments, whereas benomyl reduced disease incidence relative to the control in one trial. The study suggests that, among the five indigenous fungi, C. minitans is the most promising agent for control of white mold of dry bean under Canadian prairie conditions.     

Identity and frequency of occurrence ofTrichoderma spp. in roots of wheat and rye-grass in Western Australia and their effect on root rot caused byGaeumannomyces graminis var.tritici

Trichoderma hamatum, T. harzianum andT. koningii were isolated from wheat and rye-grass roots from a field in Western Australia. Frequency of occurrence ofTrichoderma spp. was higher on roots subjected to washing only, for both wheat and rye-grass than the roots which were surface-sterilized with 0.6% or 1.25% NaOCl.Trichoderma spp. were recovered at a higher frequency on PDA amended with lactic acid (pH 4.5) than on PDA alone (pH 5.6) or PDA with streptomycin. In general,Trichoderma spp. were isolated at a higher frequency from roots of wheat than that of rye-grass.T. hamatum occurred at a higher frequency in rye-grass roots than in wheat, whereasT. harzianum was more common in roots of wheat than in rye-grass, especially in seedling and milky ripe stages.T. koningii was recovered at a higher frequency from roots at seedling stage of rye-grass than wheat, the reverse being true at tillering stage.T. koningii was not recovered from roots of either host in any sampling when they were surface sterilized with 1.25% NaOCl.The take-all fungus was isolated from wheat and rye-grass roots more frequently at tillering and stem extension stages than others. It was severely pathogenic to both hosts in sterilized and non-sterilized soil.Addition of lactic acid, HCl or streptomycin to PDA did not affect the growth of theTrichoderma spp. tested, but the growth was slower on Martin's medium than on other media. In generalT. harzianum andT. koningii grow faster thanT. hamatum. The growth of the three species were not different at 20 and 25°C, but at 15°c growing of all species was significantly reduced.Incorporation of lactic acid into PDA prevented the bacterial growth in all treatments. Streptomycin too reduced but to a lesser degree than lactic acid. Surface sterilization with NaOCl decreased the recovery of both bacteria and fungi. T. hamatum andT. koningii reduced the mortality of wheat and rye-grass plants inoculated with the take-all fungus in sterilized and non-sterilized soil, whereT. harzianum did not protect wheat or rye-grass from infection by the take-all fungus.     

Fusarium root rot of coneflower seedlings and integrated control using <Emphasis Type="Italic">Trichoderma</Emphasis> and fungicides

Fusarium root rot (Fusarium spp.) is one of the most important seedling diseases of coneflower (Echinacea spp.) in Alberta greenhouses. Effects of microbial antagonists (Trichoderma spp.) and fungicides, including difenoconazole, fludioxonil, and a mixture of fludioxonil, metalaxyl and difenoconazole, on the management of this disease, were investigated in Alberta. Twenty Trichoderma isolates demonstrated antagonistic activity to Fusarium in agar plate bioassays, with inhibition rates ranging from 44 to 65%. Some Trichoderma isolates significantly ( p < 0.05) reduced disease incidence and severity on seedlings in greenhouse experiments. An in vitro bioassay indicated that difenoconazole and the mixture equally inhibited the growth of both Fusarium and Trichoderma, but, while fludioxonil strongly inhibited the growth of Fusarium, it had little effect on Trichoderma, according to the dose--response models developed ( p < 0.01, R²= 0.902-0.998). Two Trichoderma isolates, T1 and T13 were applied singly or in combination with a low rate of fludioxonil in greenhouse evaluations. The results suggested that fludioxonil and Trichoderma could be integrated into a disease management program for fusarium root rot in coneflower.     

Trichoderma species form endophytic associations within Theobroma cacao trichomes

Trichoderma species are usually considered soil organisms that colonize plant roots, sometimes forming a symbiotic relationship. Recent studies demonstrate that Trichoderma species are also capable of colonizing the above ground tissues of Theobroma cacao (cacao) in what has been characterized as an endophytic relationship. Trichoderma species can be re-isolated from surface sterilized cacao stem tissue, including the bark and xylem, the apical meristem, and to a lesser degree from leaves. SEM analysis of cacao stems colonized by strains of four Trichoderma species (Trichoderma ovalisporum-DIS 70a, Trichoderma hamatum-DIS 219b, Trichoderma koningiopsis-DIS 172ai, or Trichoderma harzianum-DIS 219f) showed a preference for surface colonization of glandular trichomes versus non-glandular trichomes. The Trichoderma strains colonized the glandular trichome tips and formed swellings resembling appresoria. Hyphae were observed emerging from the glandular trichomes on surface sterilized stems from cacao seedlings that had been inoculated with each of the four Trichoderma strains. Fungal hyphae were observed under the microscope emerging from the trichomes as soon as 6 h after their isolation from surface sterilized cacao seedling stems. Hyphae were also observed, in some cases, emerging from stalk cells opposite the trichome head. Repeated single trichome/hyphae isolations verified that the emerging hyphae were the Trichoderma strains with which the cacao seedlings had been inoculated. Strains of four Trichoderma species were able to enter glandular trichomes during the colonization of cacao stems where they survived surface sterilization and could be re-isolated. The penetration of cacao trichomes may provide the entry point for Trichoderma species into the cacao stem allowing systemic colonization of this tissue.     

Induced proteome of Trichoderma harzianum by Botrytis cinerea

As a notable biocontrol agent, Trichoderma harzianum can antagonize a diverse array of phytopathogenic fungi, including Botrytis cinerea, Rhizoctonia solani and Fusarium oxysporum. Elucidating the biocontrol mechanism of T. harzianum in response to the pathogens enables it to be exploited in the control of plant diseases. Two-dimensional gel electrophoresis (2-DE) was performed to obtain secreted protein patterns of T. harzianum ETS 323, grown in media that contained glucose, a mixture of glucose and deactivated B. cinerea mycelia, deactivated B. cinerea mycelia or deactivated T. harzianum mycelia. Selected protein spots were identified using liquid chromatography–tandem mass spectrometry (LC–MS/MS). Ninety one out of 100 excised protein spots were analyzed and some proteins were sequence identified. Of these, one l-amino acid oxidase (LAAO) and two endochitinases were uniquely induced in the media that contained deactivated B. cinerea mycelia as the sole carbon source. Activities of the cell wall-degrading enzymes (CWDEs), including β-1,3-glucanases, β-1,6-glucanases, chitinases, proteases and xylanases, were significantly higher in media with deactivated B. cinerea mycelia than in other media. This finding suggests that the cell wall of B. cinerea is indeed the primary target of T. harzianum ETS 323 in the biocontrol mechanism. The possible roles of LAAO and xylanase were also discussed.