Evidences of biological control capacities of Streptomyces spp. against Sclerotium rolfsii responsible for damping-off disease in sugar beet (Beta vulgaris L.)

Ten antibiotic-producing Streptomyces spp. isolated from Moroccan soils were evaluated for their ability to inhibit in vitro Sclerotium rolfsii development. Four isolates having the greatest pathogen inhibitory capabilities were subsequently tested for their ability to inhibit sclerotial germination in sterile soil. This test was carried out by using biomass inoculum, culture filtrate, and spore suspension of the isolates as treatment. Treatment with biomass inoculum and culture filtrate gave the highest inhibition of sclerotia. Biological control tests against Sclerotium rolfsii damping-off of sugar beet seeds showed that the selected Streptomyces isolates reduced significantly the disease severity, the J-2 isolate being the more potent. In addition, treatment with the isolate J-2 resulted in a significant increase (P ≤ 0.05) in seedling development compared to the control. All antagonistic Streptomyces selected here were able to grow in the rhizosphere soil from infected sugar beet culture.     

Production of transglutaminase by Streptomyces isolates in solid-state fermentation

Aims:  To screen Streptomyces isolates for transglutaminase (TGase) production in solid-state fermentation (SSF) on various substrates.
Methods and Results:  Streptomyces mobaraensis NRRL B-3729, Streptomyces paucisporogenes ATCC 12596 and Streptomyces platensis NRRL 2364 strains were screened for extracellular TGase production in SSF on different substrates. High-protein-content beans, peas and lentils proved to be the best substrates. Good TGase production was obtained on liver kidney beans and green mung beans in a 4- to 6-day SSF. Temperature optima of the enzymes varied between 45 to 50°C. Molecular weight determined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS PAGE) indicated similar size (∼37 kDa) for all three enzymes. TGase was the dominating protein band on SDS PAGE for two Streptomyces strains in SSF extracts. Other enzymes were present in smaller quantities.
Conclusions:  Streptomyces mobaraensis NRRL B-3729, S. paucisporogenes ATCC 12596 and S. platensis NRRL 2364 strains were successfully propagated under SSF conditions on crushed/milled liver kidney bean and green mung bean to obtain good level of TGase.
Significance and Impact of the Study:  Owing to much reduced production cost and direct applicability, SSF TGase without downstream processing (cheap in situ enzyme, crude enzyme) may be an excellent candidate for some nonfood applications.     

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.     

Plant growth promotion and biological control of Pythium aphanidermatum, a pathogen of cucumber, by endophytic actinomycetes

Aims:  To evaluate the potential of Actinoplanes campanulatus , Micromonospora chalcea and Streptomyces spiralis endophytic in cucumber roots, to promote plant growth and to protect seedlings and mature plants of cucumber from diseases caused by Pythium aphanidermatum , under greenhouse conditions.
Methods and Results:  Three endophytic isolates, out of 29, were selected through tests aimed at understanding their mechanisms of action as biocontrol agents and plant growth promoters. When applied individually or in combination, they significantly promoted plant growth and reduced damping-off and crown and root rot of cucumber. The combination of the three isolates resulted in significantly better suppression of diseases and plant growth promotion, than where the plants were exposed to individual strains.
Conclusions:  The three selected actinomycete isolates colonized cucumber roots endophytically for 8 weeks, promoted plant growth and suppressed pathogenic activities of P. aphanidermatum on seedling and mature cucumber plants.
Significance and Impact of the Study:  The results clearly show that the endophytic, glucanase-producing actinomycetes used, especially as a combined treatment, could replace metalaxyl, which is the currently recommended fungicide for Pythium diseases in the United Arab Emirates. These endophytic isolates also have the potential to perform as plant growth promoters, which is a useful attribute for crop production in nutrient impoverished soils.     

Thiol redox state and oxidative stress affect sclerotial differentiation of the phytopathogenic fungi Sclerotium rolfsii and Sclerotinia sclerotiorum

Aims:  To investigate the involvement of oxidative stress and thiol redox state (TRS) in sclerotial differentiation of Sclerotium rolfsii and Sclerotinia sclerotiorum.
Methods and results:  Oxidative stress in these fungi was assessed by lipid peroxidation, which was higher in comparison with their nonsclerotiogenic counterpart strains. TRS [measured as glutathione (GSH) and cysteine] was associated with oxidative stress and differentiation using the TRS modulator and antioxidant Ν -acetylcysteine (AcCSH) and the GSH biosynthesis inducer and inhibitor l -2-oxo-thiazolidine-4-carboxylate and l -buthionine- S , R -sulphoximine (BSO) respectively. Differentiation and oxidative stress was decreased by AcCSH in both fungi. The decrease of differentiation by BSO was not associated with oxidative stress in these fungi.
Conclusions:  Differentiation and oxidative stress in both fungi depends on the availability of antioxidant noncytotoxic –SH groups and is not depended on any direct antioxidant role of GSH and its precursor cysteine.
Significance and Impact of the Study:  This study helps to understand the mechanism(s) of sclerotial differentiation in these agriculturally important phytopathogenic fungi and proposes that AcCSH can be used as potent fungicide by (i) acting as growth inhibiting cytotoxic oxidant and (ii) sustaining these fungi in their undifferentiated hyphal stage where they are vulnerable to degradation by soil micro-organisms.     

Application of actinomycetes as biocontrol agents in the management of onion bacterial rot diseases

This study was conducted to achieve biological control for the post-harvest onion bacterial rot diseases with the aid of Egyptian isolates of actinomycetes. In this respect, 45 actinomycetes strains were isolated from Egyptian soils and screened for their antagonistic effect against onion bacterial rot pathogens; Erwinia carotovora subsp. carotovora and Burkholderia cepacia. The most two active strains were identified based on their cultural, morphological and molecular properties as Streptomyces lavendulae HHFA1 and Streptomyces coelicolor HHFA2, the latter was most potent and so was used in vivo (pots and field) for controlling onion bacterial rot. S. coelicolor HHFA2 application resulted in enhancement in the photosynthetic pigments and some foliar growth parameters of onion plants confirming its growth promoting effect. The results of the post-harvest estimation of the disease incidence (DI) of the onion bacterial rot throughout storage revealed that, the application of S. coelicolor HHFA2 reduced the DI pronouncedly comparing with the untreated control and confirm its successful role in the biological control of onion bacterial rot diseases.     

Inhibitory efficacy of endophytic Bacillus subtilis EDR4 against Sclerotinia sclerotiorum on rapeseed

Stem rot, caused by Sclerotinia sclerotiorum, is a serious disease of rapeseed worldwide. This paper tested the inhibitory effect of an endophytic bacterial Bacillus subtilis strain, EDR4, on the sclerotial germination and hyphal growth of S. sclerotiorum. The cell-free filtrate solution and cell suspension of strain EDR4 were sprayed on rapeseed leaves and stems one day before, at the same time and one day after inoculation in the greenhouse experiments. There was no significant difference in inhibitory efficacy between the cell-free filtrate solution and cell suspension. The best biocontrol efficacy was achieved by spraying either the cell-free filtrate solution or cell suspension at the same time as inoculation. In the field trials, the efficacy of two applications of EDR4 cell suspension at the initial flowering stage and full bloom stage was the best, but there was no significant difference in efficacy between the one-application and two-application treatments during the initial flowering stage. The efficacy decreased gradually with the culture suspension dilutions. Scanning electron microscopy revealed that EDR4 cells significantly suppressed the hyphal growth. The bacterial treatment caused shrink, cytoplasm leakage and irregular tip swelling of fungal hyphae. The hyphal cells in the treated groups had higher numbers of vacuoles in the cytoplasm than the non-treated hyphal cells. The hyphal cytoplasm was disintegrated; the hyphal biomass was reduced; the formation of infection cushions was delayed; and the infection was suppressed after spraying the bacterial culture on rapeseed leaves. The results showed that the EDR4 bacterial strain could be used to control stem rot of rapeseed.     

Plectosporium tabacinum Root Rot Disease of White Lupine (Lupinus termis Forsk.) and its Biological Control by Streptomyces Species

Plectosporium tabacinum root rot disease of white lupine is reported for the first time in Cairo Governorate provinces. Symptoms of the disease appeared on young seedlings as stunting, yellowing and wilting of the foliage. Rotted roots were first became light brown and the lesions progressively extended over the whole root system and became dark brown. Plants died when the whole root system had become infected. For successful biological control of the disease 70 rhizosphere actinomycetes were isolated by the use of normal standard isolation techniques. Three of these isolates were found to be strongly antagonistic against P. tabacinum in vitro . They also significantly reduced the incidence of white lupine root rot disease in soil infested with P. tabacinum in greenhouse trials. The three actinomycete isolates were identified as Streptomyces cyanoviridis , Streptomyces murinus and Streptomyces griseoplanus .     

Selection of antagonistic soil microbes against a few fungal pathogens of piper betle L

Summary Against the fungal pathogens,Phytophthora parasitica, Sclerotium rolfsii, Colletotrichum capsici andGlomerella cingulata responsible for leaf and foot rot of betel vines, 3 fungus, 9 actinomycetes and 4 bacterial antagonists were screened out from 61 fungus, 28 actinomycetes and 4 bacterial organisms isolated from 3 sources of soils. One each from the 3 groups of antagonists when further tested, was found quite effective against the pathogens towards neutral side of H-ion concentration. The antagonists (P. citrinum, Streptomyces sp. and bacterial organism B-7) were also found effective against 18 among 32 isolated fungus organisms from potted soil (collected from betel vine soil) and except one which happened to beP. citrinum, the rest had no adverse effect upon their growth.This work is a part of a scheme supported by Food and Agriculture Council of Pakistan and conducted in Jute Research Institute.     

Rhizoctonia solani AG 11 isolated for the first time from sugar beet in Poland

Two isolates of Rhizoctonia solani AG11 were isolated from sugar beet seedlings from South-west Poland. Both isolates gave C2 reactions in anastomose pairings with the tester isolates of AG11. The membership of both isolates to AG11 was confirmed by analysis of pectic isozyme profiles, and by verification that the internal transcribed spacer sequences of both isolates matched the references in the GenBank database. Both AG11 isolates formed white-beige to creamy-colored mycelium with wide concentric zonation. One of them formed light-colored sclerotia. The average daily rate of hyphal growth at 21 °C was 22.8 mm and 22.6 mm on PDA. They were mildly pathogenic to sugar beet seedlings due to the mycelial and secondary metabolites’ activity. The sensitivity to fungicides typically used in sugar beet protection was different for each isolate; one of them (isolate ID11) was less sensitive to thiram than the other (isolate ID3). This article discusses the worldwide occurrence of R. solani AG11, expands the currently known host range, shows its broad world distribution in regions of moderate climate, and confirms the isolates’ low frequency.     

Phenotypic variation in Streptomyces sp. DSM 40537, a lipoteichoic acid producing actinomycete

Aims:  To reinvestigate the production of lipoteichoic acid (LTA) by the actinomycete strain Streptomyces sp. DSM 40537 (=ATCC 3351).
Methods and Results:  LTA was extracted and purified from strain Streptomyces sp. DSM 40537. The identification of the LTA was confirmed by Western blotting with a monoclonal antibody. During these studies, two stable phenotypic variants of DSM 40537 were obtained, one of which released a distinctive orange pigment. 16S rRNA gene sequencing of each variant yielded identical sequences and allowed phylogenetic analysis to be performed.
Conclusions:  Streptomyces sp. DSM 40537 was shown to exhibit stable morphological variation. The strain was confirmed to be a LTA-producing actinomycete and to belong to the Streptomyces albidoflavus cluster within the genus Streptomyces .
Significance and Impact of the Study:  These data provide important support for the hypothesis that the distribution of LTA is linked to that of wall teichoic acids and emphasizes the need to reinvestigate LTA distribution in actinomycetes.     

Intergeneric conjugal gene transfer from Escherichia coli to the sweet potato pathogen Streptomyces ipomoeae

Aims:  To develop an effective gene transfer system for Streptomyces ipomoeae , the causative agent of soil rot disease of sweet potatoes.
Methods and Results:  Of the several methods investigated, introduction of genes into S. ipomoeae could only be achieved using intergeneric conjugation from Escherichia  coli . However, even results for that method varied greatly and were dependent on using particular media for S. ipomoeae spore preparation and conjugation. Transconjugant to recipient ratios as great as 4·1 × 10⁻⁵ were achieved when International Streptomyces Project Medium 4 was used for both sporulation and conjugation protocols. Both site-specifically integrating and autonomously replicating plasmids could be introduced and maintained in S. ipomoeae, and plasmids could be introduced with approximate equivalent frequencies from either methyl-proficient or methyl- deficient E. coli donors; the latter result indicates a likely absence of relevant methyl-specific restriction in S. ipomoeae .
Conclusions:  Efficient transfer of genes into S. ipomoeae was achieved here by using an optimized intergeneric mating procedure.
Significance and Impact of the Study:  The described protocol will facilitate further genetic manipulation of this agriculturally important pathogen.     

Biocontrol of Rhizoctonia crown and root rot of sugar beet by binucleate Rhizoctonia spp. and Laetisaria arvalis

Two isolates of Laetisaria arvalis and 10 of binucleate Rhizoctonia spp. (BNR) from the Ohio sugar beet production area, were tested in the greenhouse and field for biocontrol of Rhizoctonia crown and root rot of sugar beet, caused by Rhizoctonia solani anastomosis group 2, type 2. L. arvalis was ineffective in standard greenhouse tests, and the single isolate used in the field was generally ineffective. Seven of 10 BNR isolates effectively controlled crown and root rot in greenhouse tests. Delayed application of biocontrol agents to plants 5 – 10 wk old was generally more effective than applications made at planting. A BNR isolate significantly reduced % plant loss and disease ratings and increased yield in a 1985 field test as compared with the control infested with R. solani alone. Two BNR isolates were effective in a 1986 field test and increased yields c. 22% in comparison to a L. arvalis treatment, which did not differ from the R. solani-infested control. The Ohio binucleate Rhizoctonia isolates appear to have considerable potential as applied biocontrol agents and may play a role in the natural ecology of R. solani in the sugar beet production area of Ohio.     

Characterization of Iranian Pectobacterium carotovorum Strains from Sugar Beet by Phenotypic Tests and Whole-cell Proteins Profile

Thirty isolates of Pectobacterium carotovorum from soft rot‐affected sugar beet plants in the Fars province of Iran were characterized phenotypically and by analysis of whole‐cell protein electrophoresis patterns. The isolates were found to be heterogeneous based on the results of physiological and biochemical tests and protein profiles. The results of numerical analysis of phenotypic characteristics and protein patterns showed that only 27% of the collected isolates (phenon 4) could be identified as P. betavasculorum when compared with reference strains. Strains of the first, second, third and fifth phenon shared similar characters with those of P. carotovorum subsp. carotovorum, P. betavasculorum and P. carotovorum subsp. odoriferum, but were distinct from these subspecies. Inoculation of phenon 4 isolates into wounded sugar beet petioles led to black streaking, root rot and vascular necrosis. Other isolates were incapable of causing systemic symptoms in inoculated plants.     

Selective isolation,evaluation and characterization of antagonistic actinomycetes against <Emphasis Type="Italic">Rhizoctonia solani</Emphasis>

The baiting bag method was found to be useful for isolating antagonistic actinomycetes from terrestrial habitat. Out of total 110 actinomycetes isolated from rhizospheric and non-rhizospheric soil of Indo Gangetic Plains (IGP) of India, 9 isolates exhibited aggressive antagonism against Rhizoctonia solani, screened through dual culture, well diffusion and sealed plate technique. Maximum growth inhibition was recorded up to 50% under well diffusion (S. toxytricini vh22) and 52.6% in a direct confrontation (Actinomycetales bacterium vh41). Whereas maximum disease suppression (53.33%) under green house condition was achieved on seedling treated with S. tricolor vh85. Scanning electron microscopy of antagonists and pathogen interaction exhibited pore formation and hyphal degradation of test pathogen. Physiological and molecular characterization of selected isolates showed wide diversity and uncommon species has been encountered through the selective isolation technique.     

根结线虫放线菌及其生物防治活性研究

从感染植物根部的根结线虫卵和雌虫中,分离得到放线菌20株。形态、细胞壁氨基酸组分和16S rRNA基因序列分析表明:分离菌株分属于链霉菌属(Streptomyces)、诺卡氏菌属(Nocardia)和假诺卡氏菌属(Pseudonocardia),其中链霉菌占80.0%。分离菌株对根结线虫Meloidogynespp.卵的平均寄生率、卵的孵化率、幼虫死亡率分别为54.1%、40.4%和26.2%。根据体外测试的结果,选择具有高致病力的3株链霉菌(1-17,2-6,9-47)和1株诺卡氏菌(5-1)进行温室番茄防效测试,其生防效率分别为31.4%、37.7%、56.4%和42.4%。     

水霉拮抗放线菌的分离、筛选与鉴定

目的:以珍珠健康养殖水体底泥为材料分离放线菌,筛选对水产动物水霉病病原菌有抗菌活性的放线菌。方法:采用稀释涂布法,选用萘啶酮酸和放线菌酮双抗平板分离获得放线菌;以黄颡鱼和湘云鲫鱼卵水霉病原菌为靶标菌,采用琼脂块法测试所分离菌株抗水霉菌活性及其稳定性;对拮抗活性强的放线菌采用形态观察和16S r DNA序列分析进行分类鉴定。结果:从分离获得的27株放线菌中筛选出3株对水霉病原菌有拮抗活性的菌株QF1、DNC17和QHV2,其中QHV2抗菌活性与稳定性最好;形态学观察与16S r DNA序列分析结果表明QF1、DNC17和QHV2均属于链霉菌属(Streptomycete sp.),分别鉴定为Streptomyces diastatochromogenes、Streptomyces variabilis和Streptomyces collinus。结论:3株放线菌对水霉病原菌具有较好的拮抗活性,具有开发成抗水霉药物的潜在价值。     

The influence of soil-applied fungicides and previous cropping on the development of violet root rot of sugar beet

The proportion of sugar-beet roots infected by Helicobasidium purpureum increased most rapidly in September and October. Violet root rot was not controlled by fungicides applied at drilling or in July. Heavily infected roots yielded 31% less sugar than healthy or lightly infected roots. Sugar beet following infected carrots lifted or ploughed in during July had no more violet root rot than when following barley or fallow, but the beet crop became heavily infected when it followed carrots left in the ground until December, whether they were then lifted or ploughed in. Eight varieties of sno-ar beet did nnt differ in siiscenrihilitv to violet root rot.     

Bodenbewohnende Schädlinge und Seitenwurzelfäule der Zuckerrübe in Abhängigkeit von der Rotation – Ergebnisse eines Fruchtfolgeversuches

Soil inhabiting pests and rot of feeding roots of sugar beet depending on rotation - Results of a long-term trial Over a period of 17 years a trial was carried out with sugar beet, cereals and oilseed rape in different crop rotations on a field near Göttingen (Lower Saxony). The frequency of sugar beet in the rotation was 17, 25, 33 and 67 %. In absence of beet nematodes, root and sugar yield of the beet decreased after repeated growing of sugar beet in short rotations compared to variants with long rotations. Sugar content and beet quality were only slightly influenced. By applying a bioassay (BW-Test) with young beet plants in the greenhouse it was shown that increasing infections on the tips of rootlets of the beet plants were the cause for decreasing beet yield in close rotations. Mortality of young beet plants and progress of infection in the test indicated roughly the quantity of pathogenic fungi in the soil. In the roots of the bait platits the parasitic fungus Aphanomyces cochlioides predominated. Rate of infection and yield reduction in the field were decisively influenced by weather conditions. Differences in yield between sugar beet grown in a three-year and a four-year rotation, however, were not significant. An occurrence of beet pests depending on crop rotation was stated only for Atomaria linearis and this only in a few years.     

Biocontrol activity of salt tolerant Streptomyces isolates against phytopathogens causing root rot of sugar beet

Biological control of fungi causing root rot on sugar beet by native Streptomyces isolates (C and S2) was evaluated in this study. The dry weight and colony forming unit (CFU) of S2 and C increased when 300 mM NaCl was added to medium. The in vitro antagonism assays showed that both isolates had inhibitory effect against Rhizoctonia solani AG-2, Fusarium solani and Phytophthora drechsleri. In dual culture, Streptomyces isolate C inhibited mycelial growth of R. solani, F. solani and P. drechsleri 45%, 53% and 26%, respectively. NaCl treatment of medium increased biocontrol activity of soluble and volatile compounds of isolate C and S2. After salt treatment, growth inhibition of R. solani, F. solani and P. drechsleri by isolate C increased up to 59%, 70% and 79%, respectively. To elucidate the mode of antagonism, protease, chitinase, beta glucanase, cellulase, lipase and α-amylase activity and siderophore and salicylic acid (SA) production were evaluated. Both isolates showed protease, chitinase and α-amylase activity. Also, biosynthesis of siderophore was detectable for both isolates. Production of siderophore and activity of protease and α-amylase increased after adding salt for both isolates. In contrast, chitinase activity decreased significantly. Production of SA, beta glucanase and lipase by isolate S2 and biosynthesis of cellulase by isolate C were observed in presence and absence of NaCl. Soil treatment with Streptomyces isolate C inhibited root rot of sugar beet caused by P. drechsleri, R. solani and F. solani. Results of this study showed that these two Streptomyces isolates had potential to be utilized as biocontrol agent against fungal diseases especially in saline soils.