Integrated control of Rhizoctonia solani and fusarium solani in tobacco transplants with Trichoderma hurziunum and triadimenol

Trichoderma cultures were isolated locally from soils and screened on potato-dextrose agar against Rhizoctonia solani. Several isolates of T. harzianum reduced the growth and build up of populations of R. solani and to a lesser extent of Fusarium solani in sterilized soil. In field experiments, biological control of R. solani and F. solani infections in tobacco transplants was achieved by adding T. harzianum (T77) to methyl-bromide-fumigated seedbeds before seed was sown. There was also increased growth and leaf yield that was not directly correlated with disease control. Triadimenol fungicide, integrated with the Trichoderma treatment, enhanced disease control.     

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 .     

木霉菌对黄瓜生理特性及立枯病防治效果的影响

黄瓜立枯病是由立枯丝核菌（Rizoctonia solani,简称Rs）侵染引起的苗期土传病害。本研究采用前期筛选获得的对黄瓜立枯病菌Rs具有较强拮抗作用的木霉菌,分别为棘孢木霉Trichoderma asperellum 437、哈茨木霉T.harzianum 670和752,通过盆栽试验和田间试验,测定对黄瓜幼苗的生长和生理特性、产量、品质以及立枯病防效的影响。结果显示：3株木霉菌均能显著促进黄瓜幼苗的生长,提高黄瓜幼苗的生理活性和保护性酶活性,对黄瓜立枯病的防效均在75%以上;木霉菌处理的黄瓜幼苗株高、茎粗、叶面积、根体积、全株鲜重、叶绿素含量、硝酸还原酶活性、根系活力、根系总吸收面积,以及抗逆性酶活性包括超氧化物歧化酶、过氧化物酶、过氧化氢酶、多酚氧化酶均显著高于对照。木霉与病原菌Rs共同处理的黄瓜幼苗,上述生长和生理生化指标均显著高于病原菌Rs单独处理。其中木霉670处理的黄瓜幼苗各项检测指标高于木霉437和752。此外,木霉菌处理的黄瓜单瓜重、产量、可溶性固形物含量、可溶性糖含量、Vc含量、可溶性蛋白含量均不同程度的增加。上述结果说明,本研究的3株木霉菌通过提高黄瓜幼苗生理特性和保护性酶活性,促进了幼苗生长,提高了对黄瓜立枯病的抗性,改善了黄瓜产量和品质。     

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

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

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。     

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

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

木霉对玉米纹枯病的生物防治

为明确木霉菌在玉米纹枯病防治上的作用,研究了从雅安郊区大田玉米根际土壤中分离到的18个木霉菌株对立枯丝核菌的抑制作用及对玉米纹枯病的田间防治效果.对峙培养结果表明,这些木霉菌株对立枯丝核菌的抑制率为39.44%～84.98%;而木霉发酵滤液的抑菌作用为13.48%～85.81%.对峙培养和液体发酵滤液的抑菌测定相结合,筛选出木霉菌株T8、T2、T13,经鉴定分别为哈茨木霉Trichoderma harzianum、黄绿木霉Trichoderma aureoviride和长枝木霉Trichoderma Longibrachiatum.用这3种木霉的固体培养物作进一步的田间防治试验,防治效果分别为62.75%、64.48%、68.52%,比井冈霉素(49.68%)效果高;T13菌株的发酵滤液的防治效果仅为40.07%.3株木霉固体培养物对玉米产量影响显著,增产率依次为29.77%、43.37%、54.21%.     

Increased Antifungal Activity of Trichoderma harzianum Transformants That Overexpress a 33-kDa Chitinase

ABSTRACT Transformants of the biocontrol agent Trichoderma harzianum strain CECT 2413 that overexpressed a 33-kDa chitinase (Chit33) were obtained and characterized. Strain CECT 2413 was cotransformed with the amdS gene and its own chit33 gene under the control of the pki constitutive promoter from T. reesei. Southern blotting indicated that the chit33 gene was integrated ectopically, mostly in tandem. Some transformants showed the same restriction pattern, indicating preferable sites of integration. There was no correlation between the number of integrated copies and the level of expression of the chit33 gene in the transformants. When grown in glucose, the extracellular chitinase activity of the transformants was up to 200-fold greater than that of the wild type, whereas in chitin, the activity of both the transformants and the wild type was similar. Under both conditions, the transformants were more effective in inhibiting the growth of Rhizoctonia solani as compared with the wild type. Similar results were obtained when culture supernatants from the transformants and the wild type were tested against R. solani.     

Influence of methyl bromide soil fumigation method on fungicidal efficacy and bromide residues

Highest inorganic bromine residues (30 ppm) were found when soil was fumigated with liquid methyl bromide (MB) introduced by conventional means into evaporating dishes. With preheated (vaporized) MB or injection of MB/chloropicrin (CP) mixtures, bromide concentrations were reduced by 50%. They were uniform throughout the soil (0 to 60 cm) except after MB/CP injection, when larger residues were found in the 30-60 cm layer. Leaching with 2000 m^p3/ha (20 cm) of water always reduced bromide content to 7.5 and 10 ppm at the 0-30 and 30-60 cm depth, respectively. Organic amendments to soils substantially increased bromide levels up to 118 ppm, most of which was found in the upper soil layers; two teachings with 2000 m^p3 /ha water were required to return the soils to their normal state.Sclerotium rolfsii andFusarium oxysporum f. sp.dianthi cultures buried in soil were eliminated from the upper 30 cm with MB applied either conventionally or preheated. At 50 cm, 500 kg/ha of the preheated gas was superior to 1000 kg/ha of the cold gas. All MB fumigations suppressed carnation flower yields compared with CP alone but were superior to no treatment. After leaching, MB-fumigated soils yielded the highest number of flowers.     

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。     

Induction of Terpenoid Synthesis in Cotton Roots and Control of Rhizoctonia solani by Seed Treatment with Trichoderma virens

ABSTRACT Research on the mechanisms employed by the biocontrol agent Trichoderma virens to suppress cotton (Gossypium hirsutum) seedling disease incited by Rhizoctonia solani has shown that mycoparasitism and antibiotic production are not major contributors to successful biological control. In this study, we examined the possibility that seed treatment with T. virens stimulates defense responses, as indicated by the synthesis of terpenoids in cotton roots. We also examined the role of these terpenoid compounds in disease control. Analysis of extracts of cotton roots and hypocotyls grown from T. virens-treated seed showed that terpenoid synthesis and peroxidase activity were increased in the roots of treated plants, but not in the hypocotyls of these plants or in the untreated controls. Bioassay of the terpenoids for toxicity to R. solani showed that the pathway intermediates desoxyhemigossypol (dHG) and hemigossypol (HG) were strongly inhibitory to the pathogen, while the final product gossypol (G) was toxic only at a much higher concentration. Strains of T. virens and T. koningii were much more resistant to HG than was R. solani, and they thoroughly colonized the cotton roots. A comparison of biocontrol efficacy and induction of terpenoid synthesis in cotton roots by strains of T. virens, T. koningii, T. harzianum, and protoplast fusants indicated that there was a strong correlation (+0.89) between these two phenomena. It, therefore, appears that induction of defense response, particularly terpenoid synthesis, in cotton roots by T. virens may be an important mechanism in the biological control by this fungus of R. solani-incited cotton seedling disease.     

草莓根腐病防控生物药剂筛选与田间防治效果验证

草莓根腐病是陕西关中地区草莓生产中的主要病害之一。采用组织分离法对引起草莓根腐病的病原菌进行分离,通过形态学和分子生物学的方法鉴定其为茄腐镰刀菌Fusarium solani和尖孢镰刀菌Fusarium oxysporum。采用菌丝生长速率法测定了4种植物源农药对上述两种镰刀菌的抑菌活性。结果表明,1.5%苦参 ? 蛇床子素水剂(AS)对尖孢镰刀菌的抑菌活性最强,EC₅₀值为2.7 μg/mL,0.3%苦参碱乳油(EC)对茄腐镰刀菌的抑菌活性最强,EC₅₀值为4.5 μg/mL。选择室内抑菌活性较高的3种植物源杀菌剂和3种微生物菌剂 (枯草芽孢杆菌、多粘类芽孢杆菌和哈茨木霉菌) 为供试药剂进行田间试验,发现1000亿芽孢/g枯草芽孢杆菌可湿性粉剂(WP) 600倍液对草莓根腐病的防治效果最好,达69.25%,10亿芽孢/g多粘类芽孢杆菌 WP 800倍液、1.5%苦参 ? 蛇床子素 AS 700倍液、2亿芽孢/g哈茨木霉菌 WP 200倍液防治效果分别为63.14%、62.84%和59.74%。因此推荐使用枯草芽孢杆菌 WP、多粘类芽孢杆菌 WP、苦参 ? 蛇床子素 AS 和哈茨木霉菌 WP 作为草莓根腐病的田间防治药剂。     

木霉对5种病原菌抑菌活性筛选

采用滤纸片法对云南地区分离并鉴定到的15株木霉进行2株病原细菌(大肠杆菌和金黄色葡萄球菌)和3株植物病原真菌(小麦全蚀、番茄早疫、稻疫)抗菌活性筛选。获得可抑制5种病原菌(广谱)的木霉7株。抑菌圈直径范围在0.7～2.8 cm。其中哈茨木霉8917提取物对3株植物病原真菌的抑制最强,哈茨木霉10176提取物对金黄色葡萄球菌的抑制最强,哈茨木霉10174提取物对大肠杆菌的抑制最强。同时,将15株供试木霉与3株病原真菌进行对峙培养,结果显示:对番茄早疫菌抑制率最高的是新康氏木霉8722和深绿木霉8956,对小麦全蚀菌抑制率最高的是新康氏木霉8722和康氏木霉8701,深绿木霉8686对稻疫菌的抑制率最高。哈茨木霉在滤纸片法活性筛选中相对其他木霉抑菌活性较好,但在对峙培养活性筛选中其抑菌活性最弱。     

木霉不同施用方式对黄瓜膜脂过氧化作用、保护性酶活性及枯萎病防效的影响

采用3株对黄瓜枯萎病菌有较好拮抗作用的木霉,棘孢木霉Trichoderma asperellum 525、哈茨木霉T.harzianum 610和拟康氏木霉T.pseudokoningii 886,研究木霉不同施用方式对黄瓜幼苗叶片膜脂过氧化作用、保护性酶活性及防治枯萎病效果的影响。结果显示,所有木霉处理均能够提高黄瓜幼苗的株高和茎粗,增加幼苗地上部和地下部鲜重,对黄瓜枯萎病的防效均达到64%以上。播种后8~14 d,各处理的黄瓜幼苗叶片质膜透性和丙二醛(MDA)含量不同程度下降,保护性酶活性不同程度的提高,其中以先接种拟康氏木霉886,2 d后接种病原菌的T886-F处理的提高程度最显著。播种后14 d,该处理的叶片质膜透性和MDA含量比只接种病原菌的F处理分别下降了37.02%和14.80%,同时,叶片的SOD、POD、CAT、PPO活性分别比F增加了46.82%、34.93%、18.75%和11.63%。以上结果说明,本研究的3株木霉均能通过改善黄瓜幼苗叶片膜脂过氧化能力,增加保护酶活性,促进幼苗生长,防治黄瓜枯萎病。在实际应用中,提前施入木霉菌剂,有利于促进黄瓜的生长、提高幼苗抗病性、提高病害防治效果。本研究结果为木霉菌剂的高效使用、保障黄瓜安全生产提供指导。     

枸骨内生真菌抗菌代谢产物的鉴定及活性研究

对一株枸骨内生真菌哈茨木霉Trichoderma harzianum抗菌活性成分的分离、鉴定及其抗菌生物活性进行了研究。该真菌发酵液经乙酸乙酯萃取、正相硅胶和ODS-反相色谱分离纯化得到一单端孢霉烯化合物,经红外、质谱和核磁分析,鉴定为:4β-乙酰基-12,13-环氧-9-单端孢霉烯(trichodermin),这是首次从哈茨木霉菌代谢产物中分离出该化合物。Trichodermin对蕃茄早疫病和黄瓜立枯病病原菌的离体抑制活性EC50值分别为3.35和3.59 mg/L;活体测定结果表明,在 100 mg/L的剂量下对两种病的保护效果分别为97.8% 和98.1%,治疗效果为96.7%和97.3%。     

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.     

农杆菌介导的哈茨木霉T-DNA转化系统优化及拮抗能力突变子的性质分析

采用单因子试验方法研究了农杆菌EHA105介导的哈茨木霉Th-33转化过程中,各主要因素对转化效率的影响,建立了高效的转化系统,使农杆菌转化哈茨木霉的效率达到60～150个转化子/10^6个木霉孢子,利用该转化系统构建了含有8000多个转化子的T-DNA插入突变库。通过转化子与立枯丝核菌的对峙试验,从1260株转化子中筛选到23株拮抗能力发生变化的突变子。随机挑选5株突变子对其遗传稳定性进行分析,表明5株突变子都具有稳定性,聚合酶链式反应（PCR）表明上述突变子均有T-DNA片段的插入。     

黄绿木霉发酵液对水稻纹枯病菌作用的研究

利用黄绿木霉菌发酵液处理水稻纹枯病丝核菌,初步探讨了黄绿木霉菌发酵液对水稻纹枯病丝核菌的抑菌能力及抑菌机理。试验结果表明：黄绿木霉菌发酵液抑菌作用稳定,经121 ℃处理25 min后,抑菌率为100%; pH3～8的范围内抑菌率均为100%。经黄绿木霉菌发酵液处理的水稻纹枯病丝核菌菌丝电导率与呼吸强度几乎下降为0,光学显微镜观察到菌体形态发生变化,透射电镜下观察到细胞壁出现孔洞。用处理后的水稻纹枯病丝核菌菌丝接种水稻植株,发病率明显降低。     

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

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

Effect of inoculum density and soil tillage on the development and severity of rhizoctonia root rot

Rhizoctonia spp. cause substantial yield losses in direct-seeded cereal crops compared with conventional tillage. To investigate the mechanisms behind this increased disease, soils from tilled or direct-seeded fields were inoculated with Rhizoctonia spp. at population densities from 0.8 to 250 propagules per gram and planted with barley (Hordeum vulgare). The incidence and severity of disease did not differ between soils with different tillage histories. Both R. solani AG-8 and R. oryzae stunted plants at high inoculum densities, with the latter causing pre-emergence damping-off. High inoculum densities of both species stimulated early production of crown roots in barley seedlings. Intact soil cores from these same tilled and direct-seeded fields were used to evaluate the growth of Rhizoctonia spp. from colonized oat seeds. Growth of R. oryzae was not affected by previous tillage history. However, R. solani AG-8 grew more rapidly through soil from a long-term direct-seeded field compared to tilled soils. The differential response between these two experiments (mixed, homogenized soil versus intact soil) suggests that soil structure plays a major role in the proliferation of R. solani AG-8 through soils with different tillage histories.