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.     

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。     

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.     

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%.     

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。     

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.     

Effect of soil moisture on activity and dynamics of Rhizoctonia solani and Trichoderma harzianum

Journal of Plant Diseases and Protection - The effect of soil moisture on activity and dynamics of the soilborne pathogen Rhizoctonia solani and of its antagonist Trichoderma harzianum were studied...     

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。     

Control of bottom rot disease of lettuce (Rhizoctonia solani) using preparations of Trichoderma viride, T. harzianum or tolclofos-methyl

Isolates of Trichoderma were tested for their ability to control Rhizoctonia solani in lettuce seedlings and mature plants in a glasshouse. The best isolate (T. viride IMI 298375) was tested for its ability to control bottom rot disease in mature lettuce plants grown in polythene tunnels. Control was achieved in several trials but there was little evidence for much improvement after repeated applications and control with Trichoderma was poorer than that obtained with a standard treatment with tolclofos-methyl. Evidence was obtained to suggest however that enhanced growth of lettuce plants following treatment with Trichoderma can produce marketable yields of lettuce similar to those obtained with tolclofos-methyl.     

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.     

哈茨木霉几丁质酶诱导及其对水稻纹枯病菌的拮抗作用

 在实验室条件下分别以几丁质和水稻纹枯病菌(Rhizoctonia solani)细胞壁作唯一碳源诱导哈茨木霉(Trichoderma harzianum)菌株NF9、TC3和P1产生几丁质酶,用硫酸铵沉淀法制备几丁质酶粗提液。上述木霉菌株内切几丁质酶活性(对胶体几丁质浑浊度的减少率)分别为79.8%、74.4%和76.0%,均显著高于非诱导的阳性对照。培养第5 d几丁质诱导的木霉菌株NF9和TC3内切几丁质酶活性显著高于由水稻纹枯病菌细胞壁诱导的酶活性。体外测定表明,通过诱导的木霉菌株TC3、NF9和P1几丁质酶粗提液对水稻纹枯病病菌的拮抗圈直径可达38、21和23 mm,与非诱导的阳性对照比较有显著性差异。对木霉几丁质酶拮抗作用的特点及生防应用进行了讨论。     

Development of UV-induced Carbendazim-resistant mutants of Trichoderma harzianum for integrated control of damping-off disease of cotton caused by Rhizoctonia solani

Journal of Plant Diseases and Protection - The treatment of seeds with Trichoderma harzianum has been used successfully to control damping-off of cotton and other crops. However, T. harzianum is...     

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.     

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。     

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%。     

Infection of potato and wheat by isolates of Rhizoctonia solani and R. cerealis

Isolates of Rhizoctonia were obtained from potato crops with stem canker or black scurf and from cereal crops with sharp eyespot. Those with many nuclei per cell, wide cells, darkening colonies and fast growth were assigned to R. solani ; those with two nuclei per cell, narrow cells, pale colonies and slow growth were assigned to R. cerealis . Only R. solani was obtained from potatoes and only R. cerealis from cereals. On young plants in the glasshouse, the isolates of R. solani infected potato substantially but not wheat; R. cerealis infected wheat substantially and potato slightly. This host preference was shown at temperatures between 10 and 25°C in growth rooms. R. solani on potato caused more disease with increasing temperature; no trend with temperature was observed for R. cerealis on wheat.     

哈茨木霉拌种对冬小麦生长、土传病害及根际真菌群落的影响

为了明确哈茨木霉LTR-2拌种处理冬小麦的田间效果,为木霉拌种剂的推广应用提供依据,本试验从2016年－2018年连续3年,研究了哈茨木霉LTR-2拌种对小麦出苗率、幼苗生长、小麦纹枯病和茎基腐病发生情况和产量的影响,通过高通量测序和FUNGuild预测分析了木霉拌种对小麦根际土壤中真菌群落组成的影响。结果表明,哈茨木霉LTR-2拌种可以提高小麦的出苗率和冬前分蘖数;对小麦纹枯病的平均防效60%以上;对小麦茎基腐病的平均防效65%以上,优于6%戊唑醇悬浮种衣剂;与不拌种对照相比,哈茨木霉LTR-2拌种处理增产4.3%~6.34%,增产效果略高于6%戊唑醇悬浮种衣剂;木霉拌种可以降低小麦根际土壤中病原真菌的相对丰度,特别是土壤中镰孢属真菌的相对丰度。因此,哈茨木霉LTR-2可以作为化学拌种剂的绿色替代产品用于小麦生产。     

A One-Step, Immunochromatographic Lateral Flow Device Specific to Rhizoctonia solani and Certain Related Species, and Its Use to Detect and Quantify R. solani in Soil

ABSTRACT A murine hybridoma cell line GD2 secreting an immunoglobulin (Ig)M monoclonal antibody (MAb) was produced against surface antigens from an anastomosis group (AG) 4 isolate of Rhizoctonia solani (teleomorph: Thanatephorus cucumeris). Ascites were produced in mice using GD2 hybridoma cells and used to develop a rapid immunochromatographic lateral flow device (LFD) for the detection of antigens from R. solani and certain related Rhizoctonia spp. The LFD was tested for specificity against surface antigens from related and unrelated soil fungi. Antigens from representative isolates of R. solani AGs 1, 2-1, 2-3, 2-t, 3, 4, 5, 6, 7, 8, 9, 10, 11, and BI gave a positive response in LFD tests, as did antigens from Thanatephorus orchidicola, T. praticola, R. fragariae (teleomorph: Ceratorhiza fragariae), Ceratorhiza goodyerae-repentis, Ceratobasidium cornigerum, and binucleate AGE. Antigens from R. solani AGs 2-2, 2-2IIIB, and 2-2IV and from the related fungi R. carotae, R. cerealis (teleomorph: Ceratobasium cereale), R. crocorum (teleomorph: Helicobasidium brebissonii), R. oryzae (teleomorph Waitea circinata), and R. zeae gave negative responses, as did antigens from a range of unrelated fungi and oomycetes including Fusarium, Gliocladium, Trichoderma, Pythium, and Phytophthora spp. The usefulness of the LFD to detect R. solani was demonstrated in soils naturally infested with R. solani AG3. There was close agreement between results of LFD tests and conventional plate enrichment tests employing selective medium. The specificity of the technique was confirmed by polymerase chain reaction PCR using R. solani AG3-specific primers and by analyses based on sequences of the internal transcribed spacer (ITS)1-5.8S-ITS2 rRNA-encoding regions of unrelated fungi recovered from soil samples. The LFD was used to quantify R. solani AG4 in artificially infested soil samples (chopped potato soil inoculum). Estimates of CFU per gram of soil were derived using a most-probable number technique, which was based on the presence or absence of a detectable signal in the LFD. Estimates of CFU obtained in LFD tests and those obtained in a plate-trapped antigen enzyme-linked immunosorbent assay incorporating MAb GD2 were identical (449 CFU g(-1) of soil).