Use of Green Fluorescent Protein and Image Analysis to Quantify Proliferation of Trichoderma harzianum in Nonsterile Soil

ABSTRACT One drawback of traditional methods for fungal biomass measurement is the inability to distinguish biomass of an introduced fungus from that of the indigenous microbial community in nonsterile soil. We quantified biomass of a specific fungal biological control agent in nonsterile soil using epifluorescence microscopy and image analysis of green fluorescent protein (GFP)-expressing Trichoderma harzianum (ThzID1-M3). Numbers of colony forming units on a semiselective medium were compared with biomass estimates from image analysis, after ThzID1-M3 was incubated in soil that either remained moist (-0.05 MPa) for 14 to 21 days or remained moist for approximately 5 days and then was allowed to dry to <-3.0 MPa. Recovery of significant numbers of ThzID1-M3 propagules lagged approximately 3 days behind initiation of hyphal growth. Reductions in both colony counts and biomass were observed over time when soil was allowed to dry. However, in soil that remained moist, colony counts increased over a 14- to 21-day period even though biomass declined after approximately 3 to 5 days. Our results confirm that use of GFP, along with epifluorescence microscopy, is a useful tool to distinguish active hyphal biomass, the form of the fungus that is functional for biological control, from inactive propagules such as conidia or chlamydospores that are enumerated by plate counts.     

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.     

Individual-based approach to modeling hyphal growth of a biocontrol fungus in soil

ABSTRACT Fungi in soil perform beneficial roles that include biological control of soilborne plant pathogens. However, relatively little predictive information is available about the growth and activity of fungal hyphae in soil habitats. A stochastic computer simulation model ("Fungmod") was developed to predict hyphal growth of the biocontrol fungus Trichoderma harzianum ThzID1 in soil. The model simulates a fungal colony as a population of spatially referenced hyphal segments, and is individual-based, in that records of spatial location and branching hierarchy are maintained for individual hyphal nodes. In this way, the entire spatial structure of the fungal colony (hyphal network) can be explicitly reconstructed at any point in time. Also, the soil habitat is modeled as a population of spatially referenced 1-mm(3) soil cells, allowing for the simulation of a spatially heterogeneous environment. Initial hyphal growth parameters were derived from previously published results, and the model was tested against new data derived from image analysis of hyphal biomass accumulation in soil. The ability to predict fungal growth in natural habitats will help to improve the predictability of successful myco-parasitic events in biological control systems.     

Glasshouse trials of Coniothyrium minitans and Trichoderma species for the biological control of Sclerotinia sclerotiorum in celery and lettuce

Coniothyrium minitans, Trichoderma harzianum (HH3) and Trichoderma sp. (B1) were tested for ability to control disease caused by Sclerotinia sclerotiorum in a sequence of a celery crop and two lettuce crops in the glasshouse. In control plots, over 80% of celery and 90 and 60% of lettuce in first and second crops, respectively, were infected at harvest. Only the C. minitaris treatment in the first lettuce crop decreased disease and increased marketable yield. Nevertheless, C. minitans reduced the number of sclerotia recovered at harvest in the celery and first lettuce crops and decreased sclerotial survival over the autumn fallow periods following the celery and second lettuce crop. C. minitans survived in soil for over 1 year and spread to infect sclerotia in virtually all other plots. C. minitans infected sclerotia at all times of the year but sclerotia still failed to degrade during the summer months when the soil was dry. The Trichoderma species tested had no effect on disease and almost no effect on the survival of the sclerotia. even though they could be recovered from soil for the duration of the experiments.     

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.     

拟粗壮螺旋线虫对棉花的致病力及其与棉枯萎病的复合症

 温室测定结果:拟粗壮螺旋线虫(Helicotylenchus pseudorobusius)接种量在每100cm³土壤100~1000条的条件下,接种30天后棉苗生长量比无线虫对照苗显著降低(P<0.05)。抗枯萎品种86-1在单接枯萎菌无线虫处理中不发生枯萎病,在枯萎菌(Fusarium oxysporum f. sp. vasinfectum)接种量为每克土7.5×10⁵孢子和线虫(每100cm³±500~1000条)组合处理中发病,棉苗枯萎病情指数与线虫接种量之间呈正相关(γ=0.97)。田间试验结果表明:防治线虫和枯萎病复合症用薰蒸剂棉隆每m²8~14g处理土壤比用非薰蒸性杀线虫剂克百威(呋哺丹),丙线磷(益收宝)和克线磷的效果更好。     

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 .     

Biological Control of the Root-Knot Nematode Meloidogyne javanica by Trichoderma harzianum

ABSTRACT The fungal biocontrol agent, Trichoderma harzianum, was evaluated for its potential to control the root-knot nematode Meloidogyne javanica. In greenhouse experiments, root galling was reduced and top fresh weight increased in nematode-infected tomatoes following soil pretreatment with Trichoderma peat-bran preparations. The use of a proteinase Prb1-transformed line (P-2) that contains multiple copies of this gene improved biocontrol activity in the greenhouse experiments compared with the nontransformed wild-type strain (WT). All the Trichoderma strains showed the ability to colonize M. javanica-separated eggs and second-stage juveniles (J2) in sterile in vitro assays, whereas P-2 also penetrated the egg masses. This protease-transformed line presented the same nematicidal and overall proteolytic activity as the WT in in vitro tests in which concentrated soil extracts from Trichoderma-treated soils immobilized the infective J2. However, the J2 immobilization and proteolytic activities of both P-2 and the WT were higher than those obtained with strain T-203. Characterization of the activity of all Trichoderma strains soil extracts on J2 showed that it was heat resistant and restricted to the low-molecular-weight fraction (less than 3 kDa). It is suggested that improved proteolytic activity of the antagonist may be important for the biological control of the nematodes.     

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。     

土壤中盾壳霉双标记平板计数法的建立及应用

盾壳霉是一种重要的生防菌,其在土壤中的存活数量直接关系到防治病害的效果。然而目前没有对土壤中盾壳霉直接计数的方法,构建一种简单易行的土壤中盾壳霉计数方法对研究盾壳霉在土壤中的存活动态具有重要意义。本研究利用农杆菌转化法构建了潮霉素基因和绿色荧光蛋白基因标记的双标记盾壳霉菌株,并测定转化子的生长速度、产孢量和菌核致腐能力,初步分析了该方法计数土壤盾壳霉的有效性和可行性。结果显示,潮霉素基因和绿色荧光蛋白基因可以稳定地遗传和表达,并且部分转化子生长速度、产孢量和菌核致腐能力与出发盾壳霉菌株JNCM没有显著差异。加入土壤中的盾壳霉转化子可以在含潮霉素(50μg/mL)、氯霉素(100μg/mL)和链霉素(100μg/mL)的PDA平板培养,杂菌得到充分抑制,呈现绿色荧光的盾壳霉转化子被有效检出,检出限达到2×103个/g土。本研究所构建的计数方法能有效检出施入土壤中的盾壳霉并进行活菌计数,可以用于盾壳霉JN-CM产品在土壤中的定殖、生长、繁殖和存活情况的研究。应用双标记平板计数法研究了不同温度、湿度、接种量和添加菌核等条件下盾壳霉JN-CM在土壤中的存活规律。结果显示,在含有核盘菌菌核的土壤中,盾壳霉JN-CM可以通过重寄生维持一段时间(12周)的数量增长,在长达半年左右(24周)的时间里其存活率仍然可以维持在65%左右。在不含菌核的土壤中,在一般土壤温度(10~20℃)范围内,无论土壤水分含量高低,其半年存活率也可以维持在50%左右。因此,可以预测,连续施用盾壳霉JN-CM生防制剂,可以使其数量在土壤中长期维持在一定的水平范围,达到长效防治效果。     

哈茨木霉防治茉莉白绢病效果试验

1977年从水稻叶面分离获得木霉82(T—82)经鉴定为哈茨木霉Trichoderma harzianum Rifai。用这种木霉的分生孢子浓度0.5％和0.7％,防治茉莉白绢病Sclerotium rolfsii,效果分别为97.62％和100％。本文也讨论了防治机理,以及对其它病原真菌的拮抗作用。     

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%.     

Interactions of Pratylenchus thornei and Fusarium oxysporum f. sp. ciceris on Chickpea

ABSTRACT Fusarium oxysporum f. sp. ciceris and the root-lesion nematode Pratylenchus thornei coinfect chickpeas in southern Spain. The influence of root infection by P. thornei on the reaction of Fusarium wilt-susceptible (CPS 1 and PV 61) and wilt-resistant (UC 27) chickpea cultivars to F. oxysporum f. sp. ciceris race 5 was investigated under controlled and field conditions. Severity of Fusarium wilt was not modified by coinfection of chickpeas by P. thornei and F. oxysporum f. sp. ciceris, in simultaneous or sequential inoculations with the pathogens. Root infection with five nematodes per cm(3) of soil and 5,000 chlamydospores per g of soil of the fungus resulted in significantly higher numbers of propagules of F. oxysporum f. sp. ciceris with the wilt-susceptible cultivar CPS 1, but not with the wilt-resistant one. However, infection with 10 nematodes per cm(3) of soil significantly increased root infection by F. oxysporum f. sp. ciceris in both cultivars, irrespective of fungal inoculum densities (250 to 2,000 chlamydospores per g of soil). Plant growth was significantly reduced by P. thornei infection on wilt-susceptible and wilt-resistant chickpeas in controlled and field conditions, except when shorter periods of incubation (45 days after inoculation) were used under controlled conditions. Severity of root necrosis was greater in wilt-susceptible and wilt-resistant cultivars when nematodes were present in the root, irrespective of length of incubation time (45 to 90 days), densities of nematodes (5 and 10 nematodes per cm(3) of soil), fungal inocula, and experimental conditions. Nematode reproduction on the wilt-susceptible cultivars, but not on the wilt-resistant one, was significantly increased by F. oxysporum f. sp. ciceris infections under controlled and field conditions.     

Integrated control of Rhizoctonia solani by methyl bromide and Trichoderma harzianum

Under laboratory conditions, isolate TH–203 of Trichoderma harzianum was found to be tolerant of up to 20 000 ppm methyl bromide (MB) (v/v), whereas the plant pathogen Rhizoctonia solani was susceptible to a dose of less than 9000 ppm (v/v). Exposure to sub–lethal concentrations of MB had no effect on the in vitro antagonistic ability of T. harzianum . Soil fumigation with MB at the equivalent of a commercial dose of 500 kg/ha did not reduce the population of Trichoderma in soil and allowed rapid colonization of Trichoderma to develop in the soil.
Under greenhouse conditions a combination of T. harzianum and a reduced dose of MB (equivalent to 200 kg/ha) completely controlled disease incidence of R. solani in bean seedlings compared with controls in untreated soils. Similar disease control was achieved with the recommended dose of MB. Under field conditions, the combination of 200 kg/ha MB and T. harzianum gave a significant synergistic effect on damping–off of carrot seedlings caused by R. solani , and had a similar effect on growth, yield and disease control to that of the recommended dose.
T. harzianum was also able to prevent reinfestation by R. solani in fumigated soils.     

Isolation, Selection, and Efficacy of Pochonia chlamydosporia for Control of Rotylenchulus reniformis on Cotton

ABSTRACT The reniform nematode, Rotylenchulus reniformis, is a serious threat to cotton (Gossypium hirsutum) production in the United States, causing an annual loss of about $80 million. The objective of this study was to isolate fungi from eggs of R. reniformis and select potential biocontrol agents for R. reniformis on cotton. We focused on the fungus Pochonia chlamydosporia because it suppresses root-knot and cyst nematodes and because preliminary data indicated that it was present in Arkansas cotton fields. Soil samples were collected from six cotton fields in Jefferson County, Arkansas. A total of 117 isolates of the nematophagous fungus P. chlamydosporia were obtained. In an in vitro test, 105 of the 117 isolates parasitized fewer than 15% of R. reniformis eggs, but 12 isolates parasitized between 16 and 35% of the eggs. These 12 isolates produced from 6.8 x 10(4) to 6.9 x 10(5) chlamydospores per gram of medium in vitro, and chlamydospore production was similar on rice grain and corn grain media. In two greenhouse experiments, a single application of isolate 37 (5,000 chlamydospores per gram of soil) significantly reduced the numbers of R. reniformis on cotton roots and in soil. The three isolates (37, 26, and 14) that parasitized the most eggs in vitro were also the most effective in suppressing numbers of R. reniformis and in increasing cotton growth in the greenhouse.     

生防菌哈茨木霉FJAT-9040的GFP标记及土壤定殖示踪

哈茨木霉Trichoderma harzianum FJAT-9040对茄科尖孢镰刀菌具有较强拮抗作用。为跟踪分析该菌株在土壤中的存活与定殖特性,利用PEG-CaCl2介导的原生质体转化体系,筛选获得1株荧光性状稳定的菌株FJAT-9295,该菌株在生长速率、产孢量、对酸碱度和温度的适应性及对尖孢镰刀菌的抑菌活性等方面与野生型菌株FJAT-9040无显著差异（P〉0.05）。同时,研究了菌株FJAT-9295在4种类型土壤中的定殖能力以及作物生长对该菌株在土壤中定殖的影响。结果表明：菌株在育苗土中定殖最好,其次为沙土及菜园土,黄泥土中定殖最差;种植茄子比未种植作物的土壤更有利于菌株的存活;菌株FJAT-9295在不同类型土壤中的菌落数随时间的延长均略有下降,16天后趋于稳定,维持在105 CFU/g,较初始接菌量下降了约1个数量级。     

Effects of soil inoculants on the growth,transpiration and wilt disease of chickpea

Journal of Plant Diseases and Protection - Glasshouse experiments were conducted twice to assess the effects of soil inoculants (Rhizobium sp., Glomus fasciculatum and Trichoderma harzianum) and...     

Allium White Rot Suppression with Composts and Trichoderma viride in Relation to Sclerotia Viability

ABSTRACT Allium white rot (AWR) is a serious disease of Allium spp. caused by the sclerotium-forming fungus Sclerotium cepivorum. This work has examined the effects of onion waste compost (OWC) and spent mushroom compost (SMC), with and without Trichoderma viride S17A, on sclerotia viability and AWR in glasshouse and field experiments. Incorporation of OWC into soil reduced the viability of sclerotia and the incidence of AWR on onion plants in glasshouse pot bioassays, whereas SMC or T. viride S17A only reduced incidence of AWR. In two field trials, OWC reduced sclerotia viability and was as effective in reducing AWR as a fungicide (Folicur, a.i. tebuconazole). Field application of SMC had no effect on sclerotia viability and did not control AWR. However, the addition of T. viride S17A to SMC facilitated proliferation of T. viride S17A in the soil and increased the healthy onion bulb yield. The results indicate two mechanisms for the suppression of AWR: (i) reduction in the soil population of viable sclerotia, which may be due to volatile sulfur compounds detected in OWC but absent in SMC, and (ii) prevention of infection of onion plants from sclerotia following amendment of soil with OWC, SMC, or T. viride S17A.     

Interactions Between Meloidogyne artiellia, the Cereal and Legume Root-Knot Nematode, and Fusarium oxysporum f. sp. ciceris Race 5 in Chickpea

ABSTRACT In the Mediterranean Basin, Fusarium oxysporum f. sp. ciceris and the root-knot nematode Meloidogyne artiellia coinfect chickpea. The influence of root infection (after inoculation with 20 nematode eggs and second-stage juveniles per gram of soil) by two M. artiellia populations, from Italy and Syria, on the reaction of chickpea lines and cultivars with partial resistance to Fusarium wilt (CA 252.10.1.OM, CA 255.2.5.0, CPS 1, and PV 61) and with complete resistance to F. oxysporum f. sp. ciceris race 5 (CA 334.20.4, CA 336.14.3.0, ICC 14216 K, and UC 27) was investigated under controlled conditions. In genotypes with partial resistance, infection by M. artiellia significantly increased the severity of Fusarium wilt, irrespective of the fungal inoculum density (3,000 or 30,000 chlamydospores per gram of soil), except in cultivar CPS 1 at the lower fungal inoculum density. In genotypes with complete resistance to Fusarium wilt, infection by M. artiellia overcame the resistance to F. oxysporum f. sp. ciceris race 5 in CA 334.20.4 and CA 336.14.3.0 but not in ICC 14216 K, irrespective of the fungal inoculum density, and overcame the resistance in UC 27 only at the higher inoculum density. Infection by the nematode significantly increased the number of propagules of F. oxysporum f. sp. ciceris race 5 in root tissues of genotypes with complete resistance to Fusarium wilt, compared with roots that were not inoculated with the nematode, irrespective of the fungal inoculum density, except in ICC 14216 K, in which this effect occurred only at the higher inoculum density. Reproduction of an M. artiellia population from Syria in the absence of F. oxysporum f. sp. ciceris race 5 was significantly higher than that of a population from Italy in all tested chick-pea genotypes except ICC 14216 K. However, there was no significant difference between the reproduction rates of the two nematode populations in plants infected with F. oxysporum f. sp. ciceris race 5, irrespective of the fungal inoculum density and the reaction of the genotypes to the fungus.     

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。