Baiting with Sclerotium rolfsii for selective isolation of Gliocladium virens from natural soil

A baiting technique for selective isolation of Gliocladium virens from natural soil was developed by mixing Sclerotium rolfsii —colonized sorghum grains with moist natural soil and incubating at 30 ± 5°C for 6–10 days. G. virens developed large, distinct colonies on the soil surface by colonizing S. rolfsii and was then easily isolated for use in biocontrol programmes. Trichoderma spp. were present in the soil but never developed conspicuous colonies on the soil surface, even when the soil was supplemented with large numbers of conidia.     

Formation and regeneration of protoplasts in Sclerotium rolfsii ATCC 201126

AIMS: Different cultural conditions for forming and reverting protoplasts were systematically studied to establish a rapid and efficient protocol for Sclerotium rolfsii ATCC 201126. METHODS AND RESULTS: Osmotic stabilizer, lytic enzymes and mycelial age were the main factors influencing protoplast yields. An optimized protocol involving 1-h hydrolysis of 45-h-old mycelium with Trichoderma harzianum enzymes in a 1 : 1 (w/w) biomass : enzyme ratio and 0.6 mol l-1 MgSO4 as osmotic stabilizer was designed to produce approx. 2 x 109 protoplasts per gram biomass dry weight, with 99% viability. Differences on the lytic activity between batches of commercial enzymes were clearly evidenced. Protoplast release was highly efficient showing no remaining cell wall material as witnessed by fluorescent brightener 28. Up to 26% of purified protoplasts developed into the typical filamentous form after 50 h of incubation on 0.6 mol l-1 sucrose agar media. CONCLUSIONS: The methodology herein proposed allowed a rapid, inexpensive and efficient protoplast production. Optimum yields were higher or in the order of that elsewhere reported for other S. rolfsii strains and the required lytic time was significantly shorter. Purified protoplasts successfully reverted to the filamentous morphology. SIGNIFICANCE AND IMPACT OF THE STUDY: The present research reports the former protocol for the isolation and reversion of protoplasts in S. rolfsii ATCC 201126 providing key factors to ensure optimum results. In addition, the described procedure constitutes a starting point for downstream genetic manipulation.     

Integrated Control of Sclerotium rolfsii on Groundnut in South Africa

Sclerotium rolfsii Sacc. causes disease of numerous crop plants worldwide, including groundnuts. Control of this pathogen is difficult as it produces sclerotia which overwinter in the soil to emerge as inoculum and cause disease the following season. Various chemical, biological and cultural control strategies have been suggested and implemented, some of which have reduced disease incidence in the field. No studies have yet been undertaken in South Africa to control this disease on groundnut, either chemically, biologically or by cultural practices. In this study, several strategies were investigated for the control of S. rolfsii on groundnuts. Difenoconazole was identified as a fungicide that could possibly be applied in combination with Trichoderma harzianum, a biological antagonist of S. rolfsii, above carbendazim and flusilazole, and chlorothalonil. Difenoconazole significantly reduced the growth rate of S. rolfsii but not of T. harzianum. The cultivation of infected fields with an inversion plough significantly reduced infection of groundnuts by S. rolfsii and also improved the quality of the produce, while yield was not increased. Lower plant density increased the incidence of disease in an infected field, and is therefore not considered to be a viable form of cultural control.     

Phenolic compounds of Sorghum vulgare in response to Sclerotium rolfsii infection

Abstract

Identification of individual phenolic acids of Sorghum vulgare Pers. cv. M.P. after interaction with Sclerotium rolfsii Sacc. using high performance liquid chromatograph (HPLC) showed the presence of phenolics namely tannic, gallic, ferulic, chlorogenic and cinnamic acids in varying amounts. After 72 h inoculation with S. rolfsii, a maximum amount of ferulic acid (166.6 µg g^?1 fresh wt) was present in the collar of inoculated plants, followed by leaves and roots and its level decreased gradually with time. Similarly, the presence of chlorogenic acid was traced after 48 h, while that of cinnamic acid was traced after 72 h of inoculation. Reddish-brown pigmentation at the collar region of inoculated plants was also observed along with the high content of tannic acid. Among other phenolics, the presence of gallic acid was recorded consistently and maximum accumulation (139.3 µg g^?1 fresh wt) was noticed at the zone of interaction (collar region) after 72 h of inoculation. In contrast, maximum lignin deposition was observed at collar region after 96 h of inoculation. Induction of phenolic acids in S. vulgare along with the lignin deposition and red pigmentation at collar region is considered a key biomarker in the non-host-pathogen interaction in the S. valgare–S. rolfsii pathosystem.     

Trichoderma sp. as a microbial suppressive agent of Sclerotium rolfsii on vegetables

World Journal of Microbiology and Biotechnology -     

Differentiation of the causal pathogen of onion white rot Sclerotium cepivorum isolates by using the APIZYM system

The APIZYM system of detection of enzymes was proven to be useful in the differentiation of 15 European and Egyptian isolates of S. cepivorum, the incitant of onion white rot. The tested isolates produced alkaline phosphatase, esterase (c4), esterase lipase (c8), leucine arylamidase, valinearylamidase, trypsine, α-chymatrypsin, acid phosphatase, naphthol-AS-B1-phosphohydrolase, ß-galactosidase, ß-glucutronidase, α-glucosidase, ß-glucosidase and N-acetyl-ß-glucosaminidase and did not produced lipase (c14), crystine arylamidase, trypsine, ß-glucutronidase, α-mannosidase and α-fucosidase. According to enzyme activity, isolates can be divided into four groups (G). The differences between groups were in the activity of the enzymes α-chymotrypsin and α-glucosidase. The tested European isolates and the Egyptian isolates No.6 of the pathogen were in G1 and G2; however the rest of the Egyptian isolates were in G3 and G4.     

齐整小核菌内源小核菌胶水解酶的挖掘及其功能验证

小核菌胶是一种高分子水溶性微生物胞外多糖,主要用于石油开采、食品加工、医药制造和化妆品等领域.由微生物发酵法生产的小核菌胶分子量较高,存在溶解性低、粘度高和分散性差等问题,对提取、保存和应用带来了一系列困难.研究用于改性小核菌胶分子量的降解方式对扩大小核菌胶的工业应用具有重要意义.文中以齐整小核菌Sclerotium ...     

Vermicompost-based bioformulation for the management of sugarbeet root rot caused by Sclerotium rolfsii

Vermicompost-based bioformulations of bacterial and fungal biocontrol agents were examined against sugarbeet root rot caused by Sclerotium rolfsii. The result showed that the Pseudomonas fluorescens strain Pf1 in combination with either Trichoderma asperellum strain TTH1 or Bacillus subtilis strain EPCO-16 performed better in reducing disease next to the chemical difenoconazole. Similarly, enhanced yield was observed in the same combination treatments under both pot and field conditions.     

The occurrence of sclerotium rot on Momordica charantia caused by Sclerotium rolfsii in Korea

Sclerotium rolfsii is one of the most destructive pathogens and thought to affect a broad range of plant hosts. In July 2014, an occurrence of sclerotium rot was observed on bitter melon (Momordica charantia L.) in Jinju, South Korea. The rot symptoms were most developed on stems and fruit near the soil line, and infected bitter melon plants withered and eventually died. White mycelial mats with numerous sclerotia were produced on diseased stems and fruit near the soil surface. Based on the morphological characteristics, pathogenicity tests, and DNA sequencing and phylogenetic analysis of the internal transcribed spacer (ITS) ribosomal RNA (rRNA) gene region, the causal fungus was identified as S. rolfsii Saccardo. This is the first report of sclerotium rot caused by S. rolfsii on bitter melon in Korea. The recent occurrence of sclerotium rot on bitter melon poses a potential threat to its production in Korea.     

Sclerotium rolfsii management in tomato using Aframomum melegueta,Ocimum gratissimum and Cymbopogon citratus

Tomato (Lycopersicon esculentum Mill.) is a popular fruit vegetable in Nigeria but production is low due to diseases, among the various disease problems associated with tomato production is southern blight caused by Sclerotium rolfsii. This study was carried out to evaluate the efficacy of Aframomum melegueta, Ocimum gratissimum and Cymbopogon citratus in vitro and in vivo. Infected tomato stems were obtained from the experimental plots of National Horticultural Research Institute, Nigeria. Cultural and morphological identification of the organism was done with the use of microscope. A. melegueta seeds, fresh leaves of O. gratissimum and C. citratus were used. The experiment was replicated three times, radial growth (mm) was measured for 5?days at an interval of 24?h. The in vivo experiment was conducted using the same extracts in a screen house and the experiment was laid out in triplicates in completely randomised design with the synthetic fungicide as control. Data collected were subjected to analysis of variance and significant means were separated using Fisher’s Least Significant Difference at P???0.05. The results showed that the extracts differed significantly (P???0.05) in their potential to inhibit the growth of S. rolfsii. The highest reduction (1.27?mm) in radial growth was obtained with 5% O. gratissimum extract while the least (4.63?cm) was recorded with the control. Disease severity and fruit yield varied among the treatments. The highest yield (170.6?g) was obtained on plants treated with 5% O. gratissimum extract and this was comparable with the yield (155.8?g) obtained on plants treated with funguforce.     

Introduction of some new endophytic bacteria from Bacillus and Streptomyces genera as successful biocontrol agents against Sclerotium rolfsii

Endophytic bacteria live inside plant tissues without causing disease and not only promote plant growth but can also protect plants against plant pathogens. During 2010–2011 crop years, some endophytic bacteria were collected and are then biochemically and molecularly identified (16srRNA) from bean farms of East Azarbaijan, Iran. Among these bacteria isolates, four isolates from Bacillus genera and four isolates from Streptomyces genera were selected for evaluation of their ability for biocontrol of Sclerotium rolfsii in laboratory and glasshouse conditions. Except one isolate named Streptomyces parvus, the rest of isolates could significantly inhibit mycelial growth in dual culture on PDA medium. All seven selected isolates showed significant inhibition in disease treatments in glasshouse experiments. Biological traits, such as length, wet and dry weight of roots and stems in endophytic bacterial treatment showed no differences with healthy control.     

Biochemical Investigations of Sclerotial Exudates of Sclerotium rolfsii and their Antifungal Activity

Exudates from sclerotia of two Sclerotium rolfsii isolates (one causing collar rot in Cicer arietinum, isolate VC971, and the other leaf spots in Rauvolfia serpentina, isolate VL016) were assayed for their antifungal activity against 26 fungi consisting of plant parasites as well as saprophytes. Spore germination of all the test fungi was affected by the exudates reaching 100% in some cases. Foliar spray with exudates of isolate VL016 significantly reduced disease incidence of balsam (Impatiens balsamina) powdery mildew caused by Erysiphe cichoracearum and pea (Pisum sativum) powdery mildew caused by Erysiphe pisi, under field conditions. Characterization of exudates from 25 isolates of S. rolfsii revealed pH ranging from 3.8 to 5.3 and colour from light yellow to deep yellow. Among the phenolic acids found in the exudates were tannic, gallic, caffeic, vanillic, ferulic, chlorogenic and cinnamic acids. Oxalic acid was also found in varied amounts. Among the phenolic acids, ferulic acid was found to be present at high concentration in exudates of most isolates (3.9–153.4 μg/ml). The antioxidant properties of phenolics, which generally inhibit fungal morphogenesis including spore germination along with the antifungal nature of some phenolics are chiefly attributed to the inhibitory effect of sclerotial exudates of S. rolfsii. Additionally, both the isolates VC971 and VL016 showed almost similar antifungal activities despite they are of different origin and thereby demonstrate the antifungal nature of sclerotial exudates.     

Biological control of tomato collar rot induced by Sclerotium rolfsii using Trichoderma species isolated in Bangladesh

This study evaluated three Trichoderma strains (T. harzianum TR05, T. virens TR06 and T. asperellum TR08) originating from Bangladesh as potential biological control agents against collar rot of tomato under greenhouse conditions. After seed treatment with TR05, a disease incidence of collar rot (5.36%) was lower than for TR06 (34.2%) and TR08 (20.8%). Germination percentage of tomato was highest for TR05 (90.3%). In soil treatment, inoculation with TR08 resulted in the lowest disease incidence (9.78%), and the disease incidence was statistically no different from that for TR05 (16.4%). Thus, TR05 and TR08 have potential as biological control agents of collar rot in tomato.     

Evaluation of ash from some tropical plants of Nigeria for the control of Sclerotium rolfsii Sacc. on wheat ( Triticum aestivum L.)

Eleven ash samples, from organs of nine tropical plants, were screened for their abilities to inhibit mycelial growth and sclerotial germination of a Nigerian isolate of Sclerotium rolfsii on agar and in the soil. Ten ash samples showed some activity against mycelial growth of S. rolfsii in vitro. Ash samples from Delonix regia stem wood, Mangifera indica leaf and Vernonia amygdalina leaf were most effective as each totally inhibited mycelial growth of S. rolfsii in vitro. Ocimum gratissimum leaf ash, D. regia wood ash and Musa paradisiaca flower bract ash inhibited sclerotial germination on agar. Nine ash samples protected seeds against pre-emergence rot. Ash from M. indica leaf, V. amygdalina leaf and Azadirachta indica leaf protected seedlings against post-emergence infection. Eichornia crassipes ash, which was ineffective in vitro, offered some protection to seeds in soil against pre-emergence rot. The study demonstrates potentials of ash samples from tropical plants in control of S. rolfsii on wheat. This revised version was published online in June 2006 with corrections to the Cover Date.     

Ecological management of tropical sugar beet (TSB) root rot (Sclerotium rolfsii (Sacc.) by rhizosphere Trichoderma species

Trichoderma species are collected from different location of sugarbeet growing areas of Tamil Nadu and it is effective against Sclerotium rolfsii pathogen caused by sugarbeet ecosystems. Out of thirty-one isolates of Trichoderma viride and four isolates of Trichoderma harzianum collected and tested for their antagonistic activity against S. rolfsii by dual culture technique, one isolate was found to be effective T. viride (TVB1) that recorded the maximum (73.03%) inhibition on the mycelial growth recording only 2.40 cm growth as against 8.90 cm in the control. The isolates of T. harzianum THB-1 recorded 71.19% mycelial growth reduction over control. The colonisation behaviour of T. viride (TVB1) revealed that it completely over grew on pathogen within 48 h after interaction with the pathogen, and speed of growth on pathogen was also high and it possesses a higher level of competitive saprophytic ability. The best four isolates of TVB1, TVB-2, TVB-3 and TVB31 and two isolates of T. harzianum THB-1 and THB-2 were compared with other species of Trichoderma longibrachiatum, Trichoderma reesei, Trichoderma koningii and Chaetomium globosum and tested under in vitro condition. BA of neem cake at 150 kg ha^?1 + T. viride isolate (TVB1) at 2.5 kg/ha recorded least root rot disease incidence of 17.05% which accounted for 75.37% disease reduction over control and highest recorded maximum root yield 65.73 t ha^?1 and increasing sugar content.     

Influence of the nitrogen source on the production and rheological properties of scleroglucan produced by Sclerotium rolfsii ATCC 201126

Scleroglucan production by Sclerotium rolfsii ATCC 201126 has been studied using nitrate or ammonium as nitrogen sources at several concentrations. In all the experiments carried out, both growth and production were modelled by an unstructured kinetic model using logistic and Luedeking–Piret equations for describing growth and production, respectively. The kinetic parameters for growth ( and Y_XN) and for production ( and ) were obtained by fitting the data to the model using the single-response non-linear regression technique by means of the algorithm of Marquardt coupled to a fourth-order Runge–Kutta algorithm. Biomass and scleroglucan production were higher when nitrate was the nitrogen source. Rheological properties of scleroglucan produced using nitrate as nitrogen source were studied and rheological parameters calculated, revealing similarity between this biopolymer and commercial scleroglucan.     

Effect of temperature on antagonistic and biocontrol potential of shape Trichoderma sp. on Sclerotium rolfsii

Sclerotium rolfsii is a destructive soil-borne and postharvest plant pathogen. Use of the antagonistic fungus Trichoderma sp. has been earlier reported by us to successfully control this pathogen under postharvest conditions. In the present paper we report on the effects of temperature on the growth and biocontrol potential of Trichoderma sp. on S. rolfsii. Experimental results indicated that S. rolfsii and Trichoderma sp. have different temperature optima for growth: 30–35 °C for the pathogen and 25–30 °C for the antagonist. In dual culture, Trichoderma overgrew S. rolfsii at 25 °C and 30 °C, but at 35 °C and 37 °C, S. rolfsii overgrew the colony of Trichoderma. Trichoderma produced higher concentration of fungitoxic metabolites in broth culture at higher temperatures. In bioassays using ginger slices and whole rhizomes, it has been demonstrated that Trichoderma is not very effective in suppressing S. rolfsii at temperatures above 30 °C. In light of these results, possible mechanisms of biocontrol of S. rolfsii as a postharvest pathogen has been discussed. Storage temperature has been suggested as a critical factor in biocontrol of S. rolfsii. This revised version was published online in June 2006 with corrections to the Cover Date.