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.     

A greenhouse technique to screen pigeonpea for resistance to Heterodera cajani*

Heterodera cajani is an important nematode pest of pigeonpea in India and a simple and reliable greenhouse procedure has been developed to screen pigeonpea genotypes for resistance to it. In pot experiments, white cysts of H. cajani were counted on the roots of the susceptible genotype ICPL 87 at 15, 30 and 45 days after seedling emergence in soils infested with different levels of H. cajani. The seedlings were rated for the number of white cysts per root system on a one (highly resistant, no cysts) to nine (highly susceptible, more than 30 cysts) scale. White cysts were not easy to see on wet roots but were clearly visible on slightly dried roots. Cyst counts and ratings were more uniform when roots of 30 day old seedlings were evaluated than when 15 or 45 day old seedlings were examined. Effects of different H. cajani infestation levels on the ratings were not significant although the use of higher inoculum densities (16 to 27 eggs and juveniles/cm³ soil) was effective in reducing variability. This procedure was used to screen 60 pigeonpea genotypes and all of them were rated seven or nine. Ten accessions of Atylosia spp. and Rhynchosia spp. were rated three.     

Identity and Pathogenicity of Fungi Associated with Root and Crown Rot of Soft Red Winter Wheat Grown on the Upper Coastal Plain Land Resource Area of Mississippi

Seedling stand, disease severity and fungal incidence were determined from untreated ‘Wakefield’ soft red winter wheat planted on a Leeper silty clay loam in field tests conducted at the Mississippi Agricultural and Forestry Experiment Station, Plant Science Research Center, Mississippi State University, Starkville, Mississippi during the 1996–97 and 1997–98 growing seasons. Seedling stand was reduced by 40% each year in plots established with untreated seed. Cochliobolus sativus was the most frequently isolated fungus. Fusarium acuminatum, Fusarium equiseti and Fusarium solani were the most prevalent Fusarium spp. Seven other Fusarium spp. and 23 species of other fungal genera were isolated. Pathogenicity tests with three isolates each of C. sativus, Cochliobolus spicifer, F. acuminatum, F. solani, F. equiseti, Fusarium compactum, Embellisia chlamydospora and Microdochium bolleyi were performed in test tube culture and two isolates each of C. sativus, C. spicifer, F. acuminatum, E. chlamydospora and M. bolleyi under greenhouse conditions. In test tubes and in the greenhouse, seedlings infected with isolates of C. sativus developed seedling blight, discoloration and necrosis, primarily in seminal roots and crowns. In the greenhouse, C. sativus induced lesions on the lower leaf sheath and reduced seedling height, seedling emergence, dry and fresh weight of roots and shoots. Isolates of F. acuminatum, F. solani, F. equiseti, F. compactum, E. chlamydospora and M. bolleyi induced slight to moderate orange to light‐brown discoloration of crown and seminal roots in test tubes. Cochliobolus spicifer isolates had the most pre‐emergence activity, inducing black root discoloration and root pruning of wheat seedlings and reducing seedling emergence, root fresh weight and shoot dry weight. In the greenhouse, F. acuminatum reduced seedling height, seedling emergence and root and shoot dry weights. Microdochium bolleyi and E. chlamydospora reduced fresh and dry weight of roots, plant emergence and shoot dry weight. Fusarium acuminatum and C. spicifer reduced the growth rate of wheat seedlings. All fungi evaluated showed increased disease severity compared to the untreated control. The high frequency of isolation of C. sativus from crown and root tissues can be partially explained by the dry, warm conditions during the early stages of wheat seedling development in the Upper Coastal Plain Land Resource Area of Mississippi.     

Frequency and diversity of Fusarium spp. colonizing roots of Egyptian cottons

Abstract

Fusarium species are known to play a role in several diseases of cotton including the seedling disease complex, wilt, and boll rot. Therefore, a mycoflora study was conducted in 1998 in order to identify Fusarium species found in association with cotton roots. A total of 109 samples of cotton seedlings infected with post-emergence damping-off or rotted roots of adult plants were obtained from different cotton-growing areas in Egypt. Forty-six isolates were recovered and were identified as follows: F. oxysporum (28 isolates), F. moniliforme (9), F. solani (6), F. avenaceum (2), F. chlamydosporum (1). F. oxysporum, F. moniliforme and F. solani, the dominant species, accounted for 60.9%, 19.6% and 13% of the total isolates, respectively in 1998. F. oxysporum showed the highest isolation frequency in Beharia and Minufiya while F. moniliforme showed the most isolation frequency in Minufiya and Gharbiya. F. oxysporum was one of the major taxa of the Fusarium assemblage from Giza 70. F. oxysporum showed the most frequently isolated fungus in May while F. moniliforme and F. solani were the most frequently isolated fungi in August. Isolation frequency of Fusarium spp. during July and August was significantly greater than that of April or June. This implies that cotton roots are subjected more to colonization by Fusarium spp. as plants mature. Regarding pathogenicity, of the 46 isolates of Fusarium spp. tested under greenhouse conditions, 38 isolates (82.4%) were pathogenic to seedlings of Giza 89. This study indicates that F. oxysporum and F. moniliforme are important pathogens in the etiology of cotton damping-off in Egypt.     

Developmental Regulation of Susceptibility and Tolerance to Fusarium Root Rot in Beans

The soil-borne fungus, Fusarium solani f. sp. phaseoli, attacks roots and hypocotyls of bean (Phaseolus vulgaris) plants causing a devastating disease called root and foot rot. In a study of the host-pathogen relationship it was found that young bean roots, with the radicle just emerging, were highly tolerant to the pathogen, whereas older bean seedlings, with a fully developed root system, were completely susceptible. Investigations by low-temperature scanning electron microscopy demonstrated that significantly fewer spores and hyphae were present on the root surface of young bean seedlings as compared to older ones. A similar pattern of attachment was found when bean roots were inoculated with spores of F. solani f. sp. pisi, a related pathogen causing disease on peas but not on beans. Light microscopic studies showed that F. solani f. sp. pisi did not penetrate the root but rapidly formed thick-walled resting spores on the root surface. F. solani f. sp. phaseoli on the other hand quickly penetrated the root and formed an extensive network of fungal hyphae. These results demonstrate that the ability of fungal propagules to adhere to and to penetrate host tissues are two distinct processes. Furthermore, the data indicate that young bean roots lack a surface component necessary for attachment of fungal spores which may help explain their tolerance to Fusarium root rot.     

Biological control of sheath-blight of rice in india with antagonistic bacteria

Strains of fluorescent and nonfluorescent bacteria that were isolated from rice rhizospheres of Southern India and showed antagonism towardsRhizoctonia solani were evaluated for biological control of rice sheath-blight (ShB). Efficient strains of bacteria inhibited mycelial growth ofR. solani, affected sclerotial viabilityin vitro and protected IR 20 and TKM 9 rice seedlings from infection byR. solani in greenhouse tests. Pretreatment of sclerotia in bacterial suspensions resulted in reductions in ShB lesion sizes up to 31 to 44% in IR20 and 58 to 74% in TKM 9 rice. In field plots, IR 50 and TKM 9 rice plants raised from bacterized seeds had 65 to 72% less ShB than those plants from untreated seeds.     

Influence of Rhizoctonia solani on Egg Hatching and Infectivity of Rotylenchulus reniformis

The effects of culture filtrates of Rhizoctonia solani and root exudates of R. solani-infected cotton (Gossypium hirsutum) seedlings on hatching of eggs and infectivity of females of Rotylenchulus reniformis were evaluated in an attempt to account for the enhanced nematode reproduction observed in the presence of this fungus. Crude filtrates of R. solani cultures growing over sterile, deionized distilled water did not affect egg hatching. Exudates from roots of cotton seedlings increased hatching of R. reniformis eggs over that observed in water controls. Exudates from cotton seedling roots not infected or infected with R. solani did not differ in their effect on egg hatching. However, infection of cotton seedlings by reniform females was increased in the presence of R. solani, resulting in the augmented egg production and juvenile population densities in soil observed in greenhouse studies.     

Direct Delivery of Inoculum to Shoot Tissue Interferes with Genotypic Resistance to Ralstonia solanacearum in Tomato Seedlings

Employing known susceptible and resistant genotypes and pure bacterial inoculum (0.1 OD; 10⁸ CFU/ml^?1), five different inoculation methods were tried to assess the response of tomato genotypes to Ralstonia solanacearum. This included seed‐soaking inoculation, seed‐sowing followed by inoculum drenching, or at 2‐week stage through petiole‐excision inoculation, soaking of planting medium with inoculum either directly or after imparting seedling root‐injury. Seed‐based inoculations or mere inoculum drenching at 2 weeks did not induce much disease in seedlings. Petiole inoculation induced 90–100% mortality in susceptible checks but also 50–60% mortality in normally resistant genotypes within 7–10 days. Root‐injury inoculation at 2‐week seedling stage appeared the best for early and clearer distinction between resistant and susceptible lines. The observations suggest a role played by the root system in governing genotypic resistance to the pathogen. Direct shoot inoculation is to be adopted only for selecting highly resistant lines or to thin down segregating populations during resistance breeding.     

Hydrolytic Activities of Fusarium solani and Fusarium solani f. sp. eumartii Associated with the Infection Process of Potato Tubers

The development of dry rot caused by Fusarium solani f. sp. eumartii was evaluated in susceptible (Huinkul) and resistant (Spunta) potato cultivars. Fungal proteolytic and polygalacturanase activities were measured at different days postinoculation either with the pathogenic F. solani f. sp. eumartii, isolate 3122 or with the non‐pathogenic F. solani, isolate 1042. After inoculation with the pathogenic fungus, proteolytic and polygalaturonase activities were higher in the susceptible than in the resistant cultivar. In addition, we found a correlation between the levels of proteolytic activity detected in the intercellular washing fluids with the size of the lesion area caused by F. solani f. sp. eumartii in Huinkul tubers. The action of the proteolytic activity over cell wall proteins of both potato cultivars was assayed. An extracellular potato protein with homology to proteinase inhibitors of the Kunitz family was identified as a substrate of the proteolytic activity in the susceptible cultivar. A microscopic study revealed differences between the potato genotypes in the rate of response to infection by F. solani f. sp. eumartii. In addition, the cell wall alteration caused by F. solani f. sp. eumartii in cortical cells of susceptible tubers was evaluated. The data with respect to the correlation between the course of cyto‐ and biochemical events of the two host–pathogen interactions were discussed.     

Characterising seedling and adult plant resistance to Puccinia hordei in Hordeum vulgare

A set of 113 genotypes of barley (Hordeum vulgare subsp. vulgare), along with the susceptible control genotype Gus, was tested for response to the barley leaf rust pathogen Puccinia hordei in the greenhouse (as seedlings) and field (as adult plants). The tests revealed that 68 lines carried adult plant resistance (APR), 23 lines carried uncharacterised seedling resistance (USR) and that three lines carried the seedling resistance gene Rph3. Nineteen lines lacked detectable seedling resistance and were also susceptible in the field at adult plant growth stages. The presence of marker bPb‐0837, linked to the APR gene Rph20, in 35 of the 68 lines carrying APR, suggested they carry this gene. The remaining 33 lines, which lacked the Rph20 linked marker, are likely sources of new uncharacterised APR. Pedigree analysis of the 68 lines found to carry APR revealed that 32 were related to cv. Gull and to Hordeum laevigatum; two were related to cv. Bavaria and one related to cvv. Manchuria and Taganrog, suggesting that these genotypes may be the ancestral sources of the APR carried by each.     

Plant growth promotion and suppression of <Emphasis Type="Italic">Phytophthora drechsleri</Emphasis> damping-off in cucumber by cellulase-producing <Emphasis Type="Italic">Streptomyces</Emphasis>

Phytophthora drechsleri damping-off is one of the most important diseases of cucumber (Cucumis sativus). Salinity is a serious problem for crop production and affects diversity and activity of soil microorganisms. Application of salt-tolerant biocontrol agents may be beneficial in order to protect plants against pathogenic fungi in saline soils. In this study, a total of 717 Streptomyces isolates were isolated from the rhizosphere of cucumber, out of which two isolates showed more than 70% inhibitory effect against P. drechsleri and had cellulase activity in the presence and absence of NaCl. In a greenhouse experiment, two Streptomyces isolates with the highest antagonistic activity, strains C 201 and C 801, reduced seedling damping-off of cucumber caused by P. drechsleri by 77 and 80%, respectively, in artificially infested soils. Strain C 201 increased dry weight of seedlings up to 21% in greenhouse experiments. Phylogenetic analyses of 16S rRNA gene sequence reveals that strains C 201 and C 801 are closely related to S. rimosus and S. monomycini respectively. Increased activity of polyphenol oxidase (PPO) and peroxidase (POX) enzymes in Streptomyces-treated plants proved the biocontrol-induced systemic resistance (ISR) in cucumber plants against P. drechsleri.     

Breeding and selection of Buxus for resistance to Calonectria pseudonaviculata

Box blight is a widespread disease of Buxus caused by the pathogen Calonectria pseudonaviculata. It is responsible for significant losses in nurseries, gardens and wild boxwood populations. Our goal was to maximize the efficiency of a breeding programme towards increased disease resistance. The use of artificial inoculation of young F1 seedlings with C. pseudonaviculata spores under greenhouse conditions appeared to be a reliable tool for early selection of interesting prebreeding material. Overall, the four hybrid populations screened showed a segregating behaviour between their parents when determining the percentage of diseased leaves and lesion diameter. Genotypes were also found with an increased tolerance as compared to the parental species. Approximately 50% of the seedlings had the same score for both parameters after artificial inoculation in the greenhouse and in the field. Of the seedlings that showed severe symptoms in the greenhouse, <15% showed no disease symptoms in the field. Therefore, for larger breeding programmes, we propose a two‐step selection procedure: first artificial inoculation at seedling level to eliminate all genotypes with severe symptoms and then evaluation of the remaining seedlings in the field. Using this strategy, we were able to select several genotypes in our four hybrid populations with improved resistance to C. pseudonaviculata.     

Disease in wheat genotypes naturally infected with Gaeumannomyces graminis var. tritici

Wheat genotypes consisting of seven homozygous lines and 40 segregate families were studied at two sites naturally infested with the take-all pathogen, Gaeumannomyces graminis var. tritici. The numbers of seminal and coronal roots infected with G. graminis and other root pathogens were recorded. The genotypes differed in infection with G. graminis, with little evidence of genotype × environment interactions. The incidence of G. graminis and Rhizoctonia solani was negatively associated, but the association did not greatly influence differences between wheats in infection with G. graminis. The distribution of R. solani was negatively associated with the severity of take-all at only one site. Of symptoms of infection with G. graminis, wheat genotypes differed most in incidence of deadheads, but differences were not consistent over environments, and were associated with earliness of maturity. Wheats differed more in expression of disease than in infection with G. graminis. The course of disease was deduced from associations between the incidence of pathogens and components of plant growth and yield. G. graminis was the dominant pathogen at both sites, and caused a yield loss of 0–15% at one site, and an average 62% loss at the other. More components of yield were affected where disease was most severe.     

Chemical,physical and biological control of carrot seedling diseases

In naturally infested soil containingPythium ultimum, P. acanthicum andPhytophthora megasperma, onlyP. ultimum was associated with root rot and damped-off seedlings. Damping-off was promoted by low soil temperatures and by flooding. Seedling stands were markedly reduced when seed was pre-incubated in soil at 12°C but not at 25°C or 35°C. Dusting carrot seed with metalaxyl significantly increased seedling stands in the field at rates from 1.5–6 g kg⁻¹ seed and in both flooded and unflooded, naturally infested soil at 3.15 g kg⁻¹. In greenhouse experiments using artifically infested soil,P. ultimum andP. paroecandrum caused damping-off of carrot seedlings andRhizoctonia solani reduced root and shoot weights.R. solani caused damping-off in nutrient-enriched soil.P. acanthicum andP. megasperma were not pathogenic to seedlings, although both fungi colonized roots. Soil populations of allPythium spp., particularlyP. ultimum, increased during growth of seedlings and population growth ofP. megasperma was promoted by periodic flooding. Infestation of soil withP. acanthicum did not reduce damping-off of carrot seedlings byP. ultimum orP. paroecandrum, but significantly increased root and shoot weights and decreased root colonization byR. solani P. acanthicum has potential as a biocontrol agent againstR. solani.     

In vitro and ex vitro Selection of Potato Plantlets for Resistance to Early Blight

Reaction to the culture filtrate of Alternaria solani (Sorauer) was used as an indicator in an in vitro screening test to select lines with decreased field susceptibility to early blight from a population of 1000 putative mutants. Plantlets of cv. ‘Desirée derived from irradiated callus of potato were inoculated in vitro with a culture filtrate of A. solani (Sorauer). Of the 45 lines selected and subsequently evaluated under conditions of natural infection in the greenhouse six showed lesser degrees of early blight infection than the cv. Desirée control. The six lines selected in the greenhouse retained lower degrees of infection during 2 years of field trials.     

First Report of Rhizoctonia solani AG‐7 on Cotton in Egypt

Eighty‐two isolates of Rhizoctonia solani were recorded from roots of naturally‐infected seedlings of the Egyptian cotton (Gossypium barbadense L.). Anastomosis groups (AGs) of the isolates were determined by using 13 different AGs testers. Three (3.7%) of the isolates were identified as R. solani AG7, while the remaining isolates were belonging to the AG 2‐1, AG4 and AG5. The identification of the three isolates was based on the frequency of the C2 reaction with the AG7 tester isolate. No fusion was observed between AG7 and isolates representing the other 13 AGs. Colonies of AG7 isolates grown on potato dextrose agar (PDA), malt yeast agar (MYA) and melt peptone agar (MPA) were brown to dark brown with aerial mycelium and sclerotia. The isolates had pitted sclerotial clusters and brownish exudates after 21 days of culturing on PDA, but without clear zonation. Pathogenicity test under greenhouse conditions revealed that AG7 caused the common symptoms of damping–off, which included seed rot, lesions on the hypocotyls and root rot.     

Comparison of fungicides for the control of Rhizoctonia solani causing datnping-off of mung bean (Phaseohis aureus)

Of 41 fungicides tested in the laboratory, copper carbonate, copper sulphate, mercuric chloride, Agrosan GN, quintozene, kasugamycin, carboxin, pyracar-bolid, carbendazim, chloroneb, benomyl, Ohric, RH 893 (2-n-octyl-4-isothiazole-3-one) and Terrazole were most inhibitory to the mycelial growth of Rhizoctonia solani on Czapek's agar plates and had EC₅₀ values of less than 1 μg a.i./ml, while copper oxychloride, Udonkor, zineb, ziram, F 319 (3-hydroxy-5-methyl isoxazole) and anilazine were much less toxic, ziram being least inhibitory with an EC₅₀ of 214 μg a.i./ml. Of 17 fungicides tested in the greenhouse as seed treatments, thiabendazole, carbendazim, benomyl, thiophanate-methyl, dichlozoline and Ohric gave 80–90% control of damping-off of mung bean seedlings. A single soil drench with thiophanate-methyl and two drenches with benomyl gave about 90% disease control, More seedlings with R. solani infection survived when thiophanate-methyl was used as a post-inoculation soil drench than when benomyl or chloroneb were used.     

Influence of Fusarium solani on citrus root rot caused by Phytophthora parasitica and Phytophthora citrophthora

Interactions between Fusarium solani and Phytophthora parasitica or F. solani and P. citrophthora influenced the development of root rot of citrus but depended on the temporal order of inoculation with F. solani or the two Phytophthora spp. Inoculation of citrus with either Fusarium solani and Phytophthora parasitica or Phytophthora citrophthora increased root rot compared to inoculation with P. parasitica or P. citrophthora alone when plants were inoculated with Phytophthora by dipping their roots in zoospore suspensions and subsequently transplanted into soil infested with F. solani. However, root rot was not increased by simultaneous co-inoculation of P. parasitica and F. solani or when plants were inoculated with F. solani first. Root rot was not increased when heat-stressed or non-stressed plants were inoculated with P. parasitica 30 days after transplanting into soil infested with F. solani. In most but not all experiments, F. solani alone reduced growth of tops or roots a small but significant amount.Co-inoculation of citrus by root-dipping into zoospore suspensions of P. parasitica and transplanting into soil infested with F. solani reduced feeder root length by 62% and root weight by 61% but did not significantly reduce the percentage of living roots when compared to inoculation with P. parasitica alone. When citrus roots were immersed in zoospore suspensions of P. citrophthora and transplanted into soil infested with F. solani, feeder root length was reduced by 68%, but feeder root weight and the percentage of living roots were not significantly reduced when compared to plants inoculated with P. citrophthora alone.Propagule densities of both P. parasitica and P. citrophthora in the rhizosphere of plants inoculated by root-immersion and then transplanting into soil infested with F. solani were not significantly different than propagule densities from plants transplanted into non-infested soil. Propagule densities of P. parasitica were suppressed an average of 41% when citrus was inoculated with P. parasitica 30 days after transplanting into soil infested with F. solani and by 41% when citrus was co-inoculated by transplanting into soil infested with both F. solani and P. parasitica.     

Intensity of radicle fluorescence as related to the resistance of seedlings of lettuce to the lettuce root aphid and carrot to the carrot fly

A technique is described which indicates by u.v. fluorescence the concentration of caffeoylquinic, chlorogenic and isochlorogenic acids in roots of germinating lettuce and carrot. The surface fluorescence of radicles from lettuce root aphid (Pemphigus bursarius) resistant seedlings was more intense than for susceptible seedlings, attributable to the higher concentration of isochlorogenic acid. In contrast, radicles of carrot seedlings resistant to carrot fly (Psila rosae) were less fluorescent than susceptible seedlings, corresponding to the lower concentration of chlorogenic acid in resistant seedlings. The technique is non-destructive and has been developed to distinguish between cultivars or breeding lines potentially resistant to these insect pests.