Pectic zymogram variation and pathogenicity of <Emphasis Type="Italic">Rhizoctonia solani</Emphasis> AG-4 to bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis>) isolates in Isfahn,Iran

Rhizoctonia disease, caused by Rhizoctonia solani is one of the most important fungal diseases in bean fields in Isfahan, Iran. Bean plants showing stem and root cankers were collected and Rhizoctonia-like fungi obtained from the samples were identified by anastomosis. Pure cultures of bean isolates of R. solani were identified as AG-4. There were also AG-4 isolates from tomato, potato, cucumber, alfalfa and sugar beet in the areas sampled. A total of 163 isolates of R. solani AG-4 originating from stem and root cankers of beans were examined using pectic zymogram electrophoresis. Polygalacturonase (PG) and pectin estrase isozymes were observed in all AG-4 isolates tested. One (PG) and one pectic esterase (PE) band was found in common between all isolates examined. The electrophoretic patterns were grouped into seven zymogram groups (ZGs) according to the diagnostic PG and PE bands. One ZG occurred in a high frequency throughout the areas sampled. A pathogenicity test was conducted and representative isolates of each ZG were used to inoculate healthy bean plants. The results showed that each ZG caused different symptoms with varying severity. Isolates belonging to two ZGs were highly pathogenic causing root, stem and hypocotyl cankers whereas isolates of the other ZGs produced weak or no symptoms.     

Diverse mechanisms adopted by fluorescent <Emphasis Type="Italic">Pseudomonas</Emphasis> PGC2 during the inhibition of <Emphasis Type="Italic">Rhizoctonia solani</Emphasis> and <Emphasis Type="Italic">Phytophthora capsici</Emphasis>

Rhizoctonia solani and Phytophthora capsici are two of the most destructive phytopathogens occurring worldwide and are only partly being managed by traditional control strategies. Fluorescent Pseudomonas isolates PGC1 and PGC2 were checked for the antifungal potential against R. solani and P. capsici. Both the isolates were screened for the ability to produce a range of antifungal compounds. The results of this study indicated the role of chitinase and β-1,3-glucanase in the inhibition of R. solani, however, antifungal metabolites of a non-enzymatic nature were responsible for inhibition of P. capsici. The study confirmed that multiple and diverse mechanisms are adopted by the same antagonist to suppress different phytopathogens, as evidenced in case of R. solani and P. capsici.     

Clavatol and patulin formation as the antagonistic principle of <Emphasis Type="Italic">Aspergillus clavatonanicus</Emphasis>, an endophytic fungus of <Emphasis Type="Italic">Taxus mairei</Emphasis>

Many endophytic fungi are known to protect plants from plant pathogens, but the antagonistic mechanism has rarely been revealed. In this study, we wished to learn whether an endophytic Aspergillus sp., isolated from Taxus mairei, would indeed produce bioactive components, and if so whether (a) they would antagonize plant pathogenic fungi; and (b) whether this Aspergillus sp. would produce the compound also under conditions of confrontation with these fungi. The endophytic fungal strain from T. mairei was identified as Aspergillus clavatonanicus by analysis of morphological characteristics and the sequence of the internal transcribed spacers (ITS rDNA) of rDNA. When grown in surface culture, the fungus produced clavatol (2′,4′-dihydroxy-3′,5′-dimethylacetophenone) and patulin (2-hydroxy-3,7-dioxabicyclo [4.3.0]nona-5,9-dien-8-one), as shown by shown by NMR, MS, X-ray, and EI-MS analysis. Both exhibited inhibitory activity in vitro against several plant pathogenic fungi, i.e., Botrytis cinerea, Didymella bryoniae, Fusarium oxysporum f. sp. cucumerinum, Rhizoctonia solani, and Pythium ultimum. During confrontation with P. ultimum, A. clavatonanicus antagonized its growth of P. ultimum, and both clavatol as well as patulin were formed as the only bioactive components, albeit with different kinetics. We conclude that A. clavatonanicus produces clavatol and patulin, and that these two polyketides may be involved in the protection of T. mairei against attack by plant pathogens by this Aspergillus sp.     

Antifungal activity of lichen extracts and lichenic acids

Acetone extracts of the threelichen species Evernia prunastri, Hypogymnia physodes and Cladoniaportentosa were investigated for activityagainst eight plant pathogenic fungi: Pythium ultimum, Phytophthorainfestans, Rhizoctonia solani, Botrytis cinerea, Colletotrichumlindemuthianum, Fusarium solani, Stagonospora nodorum and Ustilagomaydis. Particularly, E. prunastriand H. physodes exhibit total or stronginhibition on P. ultimum, U.maydis and P. infestans growth. Incontrast, Cladonia extracts were lessactive to reduce growth of these fungi.Lichenic acids were also examined forantifungal activity. P. infestans growthwas severely inhibited by evernic acid. P.ultimum and P. infestans growth wereslightly but significantly inhibited by evernicacid and (–) usnic acid, respectively. Growthinhibition of U. maydis was also observedfor the latter lichenic acid. These resultsconfirm the previously observed activities oflichen extracts. This suggests that secondarylichen metabolites might be of potential use asantifungal agents.     

Interaction of cruciferous phytoanticipins with plant fungal pathogens: indole glucosinolates are not metabolized but the corresponding desulfo-derivatives and nitriles are

Glucosinolates represent a large group of plant natural products long known for diverse and fascinating physiological functions and activities. Despite the relevance and huge interest on the roles of indole glucosinolates in plant defense, little is known about their direct interaction with microbial plant pathogens. Toward this end, the metabolism of indolyl glucosinolates, their corresponding desulfo-derivatives, and derived metabolites, by three fungal species pathogenic on crucifers was investigated. While glucobrassicin, 1-methoxyglucobrassicin, 4-methoxyglucobrassicin were not metabolized by the pathogenic fungi Alternaria brassicicola, Rhizoctonia solani and Sclerotinia sclerotiorum, the corresponding desulfo-derivatives were metabolized to indolyl-3-acetonitrile, caulilexin C (1-methoxyindolyl-3-acetonitrile) and arvelexin (4-methoxyindolyl-3-acetonitrile) by R. solani and S. sclerotiorum, but not by A. brassicicola. That is, desulfo-glucosinolates were metabolized by two non-host-selective pathogens, but not by a host-selective. Indolyl-3-acetonitrile, caulilexin C and arvelexin were metabolized to the corresponding indole-3-carboxylic acids. Indolyl-3-acetonitriles displayed higher inhibitory activity than indole desulfo-glucosinolates. Indolyl-3-methanol displayed antifungal activity and was metabolized by A. brassicicola and R. solani to the less antifungal compounds indole-3-carboxaldehyde and indole-3-carboxylic acid. Diindolyl-3-methane was strongly antifungal and stable in fungal cultures, but ascorbigen was not stable in solution and displayed low antifungal activity; neither compound appeared to be metabolized by any of the three fungal species. The cell-free extracts of mycelia of A. brassicicola displayed low myrosinase activity using glucobrassicin as substrate, but myrosinase activity was not detectable in mycelia of either R. solani or S. sclerotiorum.     

Antagonism against Rhizoctonia solani and fungitoxic metabolite production by some Penicillium isolates

A number of Penicillium isolates were recovered in association to Rhizoctonia solani strains pathogenic on tobacco and from soil on plates pre-colonized by the pathogen itself. Their antagonism toward R. solaniAG-2-1 was evaluated in dual cultures in vitro. Inhibition of growth was evident to some extent in most pairings, while hyphal interactions referable to mycoparasitic relationships were not observed. However, the occurrence of plasmolysis and/or vacuolisation and the induction of monilioid cells were indicative of the release of bioactive compounds. Therefore, production of fungitoxic metabolites was tested by adding concentrated culture filtrates of each Penicillium isolate to the growth medium of R. solani. Complete and lasting inhibition was incited by culture filtrates of some isolates belonging to P. brevicompactum, P. expansum, and P. pinophilum. Three purified compounds, respectively mycophenolic acid, patulin and 3-O-methylfunicone, which were extracted from culture filtrates, were able to inhibit R. solani in vitro. Their production was also detected in dual cultures of the same Penicilliumstrains with R. solani prepared in sterilized soil and when the Penicilliumstrains were cultured directly on R. solani mycelium harvested from liquid cultures. The possible role of such metabolites in antagonism of the above-mentioned Penicilliumspecies against R. solani is discussed.     

Mycoparasitism studies of Trichoderma harzianum strains against Rhizoctonia solani: evaluation of coiling and hydrolytic enzyme production

The genus Trichoderma is a potential biocontrol agent against several phytopathogenic fungi. One parameter for its successful use is an efficient coiling process followed by a substantial production of hydrolytic enzymes. The interaction between fifteen isolates of Trichoderma harzianum and the soil-borne plant pathogen, Rhizoctonia solani, was studied by light microscopy and transmission electron microscopy (TEM). Macroscopic observations of fungal growth in dual cultures revealed that growth inhibition of the pathogen occurred soon after contact with the antagonist. All T. harzianum isolates tested exhibited coiling around the hyphae of R. solani. The strains ALL23, ALL40, ALL41, ALL43 and ALL49 did not differ in coiling frequency and gave equal coiling performances. No correlation between coiling frequency and the production of cell wall-degrading chitinases, N-acetyl-β-d-glucosaminidase and β-1,3-glucanases, was found.     

林下参内生生防细菌的分离鉴定及定殖能力

【背景】多年生林下参在自然环境下生长多年,其体内存在的内生菌具有更强的适应性和定殖性,可以提高植物自身抗性,抑制病原菌的生长,更好地发挥与植物的互作。【目的】筛选定殖能力强、繁殖能力快且对病原菌具有拮抗作用的优势菌株。【方法】采用常规组织分离方法,从健康林下参根部组织中分离内生菌,通过对峙试验筛选出对人参病原菌有拮抗作用的内生细菌并对其以传统的鉴定方法进行鉴定。【结果】在得到的6株内生细菌中,菌株LXS-N2对人参立枯病病原菌、人参猝倒病病原菌均有明显抑菌性,而且具有定殖性好、繁殖快的特点,通过破坏病原真菌细胞壁和细胞膜以及改变菌丝形态从而抑制病原真菌生长。【结论】经形态学观察、生理生化反应及16S rRNA基因序列分析鉴定内生菌LXS-N2为贝莱斯芽孢杆菌,具有良好的应用开发潜力。     

Biologically Active Endophytic Streptomycetes from <Emphasis Type="Italic">Nothofagus</Emphasis> spp. and Other Plants in Patagonia

Endophytic streptomycetes have been isolated and characterized from several species of Nothofagus and other plants growing in the southern reaches of Patagonia. No endophytic streptomycete was obtained from any plant species studied in Northern Patagonia. However, from Southern Patagonia, biologically active Streptomyces spp. from several plant species were isolated. Each isolate, as studied by scanning electron microscopy (SEM), has small hyphae, some produce typical barrel-shaped spores in culture and each has some unique hyphal surface structures. Interestingly, although none has any detectable antibacterial killing properties, each has demonstrable killing activity against one or more pathogenic fungi including representative plant pathogenic organisms such as Phytophthora erythroseptica, Pythium ultimum, Sclerotinia sclerotiorum, Mycosphaerella fijiensis, and Rhizoctonia solani. The 16S rDNA sequences of the isolates were distinct from all other genetic accessions of Streptomyces in GenBank. However, isolate C-2 from Chiliotrichum diffusum (Compositae) is identical, in all respects, to isolate C-4 obtained from Misodendrum punctulatum (Loranthaceae). These results confirm that endophytic streptomycetes represent a novel source of biologically active microorganisms.     

Efficacy of benzimidazole and related fungicides against Rhizoctonia solani and R. bataticola

Five formulations of four benzimidazole derived fungicides, carbendazim, benomyl, thiophanate methyl and methyl 4-[2-(2-dimethylamino acetamide) phenyl]-3-thioallophanate were compared for their toxicity towards two pathogenic isolates of Rhizoctonia solani and three of R. bataticola. The isolates of two fungi showed significant differences in mycelial growth inhibition by the five fungicides. Benomyl and carbendazim were most inhibitory to all isolates of both fungi while the sesame isolate of R. bataticola was least sensitive to all fungicides. Disease control (90%) was obtained with low concentrations of benomyl against root rot of cowpea caused by R. solani, and with thiophanate methyl against root rot of sesame and sunflower, and leaf blight of mung bean caused by R. bataticola. The spread of stalk-end rot of sunflower heads was best checked with a spray of thiophanate methyl. The results suggest that benzimidazole fungicides having similar toxophores act differently for disease control in different host-parasite combinations.     

The feeding preference of mycophagous Collembola varies with the ectomycorrhizal symbiont

The interactions of the collembolan insect Proisotoma minuta with ectomycorrhizal and/or pathogenic fungi was examined in three experiments: (1) in vitro analysis of feeding patterns, (2) in vitro food preference test, and (3) in situ analysis of ectomycorrhizal colonization in relation to population density. The ectomycorrhizal fungi Laccaria laccata, Pisolithus tinctorius, Suillus luteus, Thelephora terrestris and the pathogenic fungi Rhizoctonia solani were employed in all experiments. In vitro and in situ experiments revealed that Pr. minuta consumed all the ectomycorrhizal fungi tested but the feeding pattern and consumption varied with each isolate. In a comparative in vitro feeding preference test, where Pr. minuta was given a choice, R. solani was grazed more heavily than the ectomycorrhizal fungi. Among the ectomycorrhizal fungi examined, Pi. tinctorius was consumed significantly less than L. laccata, S. luteus or T. terrestris in the presence of R. solani. A 10-week in situ analysis of loblolly pine (Pinus taeda L.) seedling root systems inoculated with Pr. minuta revealed that ectomycorrhizal colonization was significantly less than that of control plants (without Pr. minuta). Collectively, these data suggest that mycophagous Collembola may play a major role in the distribution and biomass of ectomycorrhizal fungi in the rhizosphere of tree seedlings.     

Microbial conversion and in vitro and in vivo antifungal assessment of bioconverted docosahexaenoic acid (bDHA) used against agricultural plant pathogenic fungi

Microbial modification of polyunsaturated fatty acids can often lead to special changes in their structure and in biological potential. Therefore, the aim of this study was to develop potential antifungal agents through the microbial conversion of docosahexaenoic acid (DHA). Bioconverted oil extract of docosahexaenoic acid (bDHA), obtained from the microbial conversion of docosahexaenoic acid (DHA) by Pseudomonas aeruginosa PR3, was assessed for its in vitro and in vivo antifungal potential. Mycelial growth inhibition of test plant pathogens, such as Botrytis cinerea, Colletotrichum capsici, Fusarium oxysporum, Fusarium solani, Phytophthora capsici, Rhizoctonia solani and Sclerotinia sclerotiorum, was measured in vitro. bDHA (5 μl disc⁻¹) inhibited 55.30–65.90% fungal mycelium radial growth of all the tested plant pathogens. Minimum inhibitory concentrations (MICs) of bDHA against the tested plant pathogens were found in the range of 125–500 μg ml⁻¹. Also, bDHA had a strong detrimental effect on spore germination for all the tested plant pathogens. Further, three plant pathogenic fungi, namely C. capsici, F. oxysporum and P. capsici, were subjected to an in vivo antifungal screening. bDHA at higher concentrations revealed a promising antifungal effect in vivo as compared to the positive control oligochitosan. Furthermore, elaborative study of GC-MS analysis was conducted on bioconverted oil extract of DHA to identify the transformation products present in bDHA. The results of this study indicate that the oil extract of bDHA has potential value of industrial significance to control plant pathogenic fungi.     

Investigation of a Great Number of Actinomycete Isolates on Their Antagonistic Effects Against Soil-Borne Fungal Plant Pathogens by an Improved Method

By means of a modified agar-ring method, the antibiotic efficacy of 300 isolates of actinomycetes, obtained from soil samples from Turkey, against six different test fungi was investigated. A wide range in their degree of sensitivity was evident. Whereas Sclerotinia sclerotiorum was completely inhibited by more than 90 % of the tested isolates, Rhizoctonia solani and Alternaria alternata were suppressed by only 17 and 14 %, respectively; Pythium debaryanum, Cochliobulus sativ, us and Macrophomina phaseolina held an intermediate position. Isolates which completely prevented the growth of Rhizoctonia solani and Alternaria alternata, were usually also highly effective against the other test fungi. Possible conclusions which may be drawn from these results are discussed.     

Study of the antifungal activity of <Emphasis Type="Italic">Acinetobacter baumannii</Emphasis> LCH001 in vitro and identification of its antifungal components

An Acinetobacter strain, given the code name LCH001 and having the potential to be an endophytic antagonist, has been isolated from healthy stems of the plant Cinnamomum camphora (L.) Presl, guided by an in vitro screening technique. The bacterium inhibited the growth of several phytopathogenic fungi such as Cryphonectria parasitica, Glomerella glycines, Phytophthora capsici, Fusarium graminearum, Botrytis cinerea, and Rhizoctonia solani. Biochemical, physiological, and 16S rDNA sequence analysis proved that it is Acinetobacter baumannii. When the filtrate from the fermentation broth of strain LCH001 was tested in vitro and in vivo, it showed strong growth inhibition against several phytopathogens including P. capsici, F. graminearum, and R. solani, indicating that suppression of the growth of the fungi was due to the presence of antifungal compounds in the culture broth. Moreover, the antifungal activity of the culture filtrate was significantly correlated with the cell growth of strain LCH001. The active metabolites in the filtrate were relatively thermally stable, but were sensitive to acidic conditions. Three antifungal compounds were isolated from the culture broth by absorption onto macropore resin, ethanol extraction, chromatography on silica gel or LH-20 columns, and crystallization. The structures of the bioactive compounds were identified by spectroscopic methods as isomers of iturin A, namely, iturin A2, iturin A3, and iturin A6. The characterization of an unusual endophytic bacterial strain LCH001 and its bioactive components may provide an alternative resource for the biocontrol of plant diseases.     

Suppression by Zygorrhynchus moelleri of Growth and Sclerotium Production by Rhizoctonia solani and Sclerotinia sclerotiorum

As indicated by reduced cellulolysis, Zygorrhynchus moelleri suppressed mycelial growth in Rhizoctonia solani and Sclerotinia sclerotiorum. Sclerotium production by both pathogenic fungi was also reduced by Z. moelleri in dual sand-oatmeal cultures. The viability of sclerotia produced by S. sclerotiorum, but not those produced by R. solani, was greatly reduced. Sclerotium production by S. sclerotiorum on celery and tomato segments was reduced to a much greater extent when Z. moelleri was applied to the plant tissue 24 h before the pathogen than when applied at the same time or 24 h after the pathogen.     

Effect of kind and method of fungicidal treatment of bean seed on infections by the VA-mycorrhizal fungus Glomus macrocarpum and by the pathogenic fungus Fusarium solani

To test the effect of seed treatment with fungicides on the development of mycorrhizal fungi, bean seeds were treated with fungicide dry or vehicled in the organic solvents, ethanol or dichloromethane and then planted in soil inoculated with the mycorrhizal fungus Glomus macrocarpum and/or the plant pathogenic fungus Fusarium solani. Measurements were made at 4 day intervals, to evaluate the location and extent of colonization of either Glomus macrocarpum or Fusarium solani in the root system. Most combinations of fungicide-solvent had little effect on the extent of colonization by each fungus individually. However, when both fungi were inoculated together, symptoms of F. solani were seen only in the tips of roots which indicate that the mycorrhizal fungus was able to limit the occurrence of the pathogenic fungus.     

Antimycotic activities of Cinnamon-derived compounds against <Emphasis Type="Italic">Rhizoctonia solani</Emphasis> in vitro

In this study, the effects of medicinal plant extracts on the development of mycelium in the following phytopathogenic fungi were evaluated: Phytophthora capsici, Rhizoctonia solani, Fusarium solani, Colletotrichum gloeosprorioides, and Botrytis cinera. Of the 26 medicinal plants tested, six plant extracts showed antifungal activity against phytopathogenic fungi. The highest antifungal activity was exerted against R. solani by the n-hexane fraction of a Cinnamon (Cinnamomum cassia Blume) solvent extract. Therefore, the antifungal compound fractions I and II were purified from the n-hexane fraction by TLC on silica gel plates. When treated with solutions containing compound fractions I or II at a concentration of 2%, the mycelia growth rate of R. solani was reduced to 0.19 and 0.18, respectively. In addition, microscopic observation of the hyphal morphology of R. solani following treatment with compound fraction I revealed the presence of severely damaged hyphae. Specifically, the hyphal tips became swollen, collapsed or were completely destroyed in response to treatment with solution containing compound fraction I at concentration of 1%.     

Induction of pathogenesis-related proteins during biocontrol of <Emphasis Type="Italic">Rhizoctonia solani</Emphasis> with <Emphasis Type="Italic">Pseudomonas aureofaciens</Emphasis> in soybean (<Emphasis Type="Italic">Glycine max</Emphasis> L. Merr.) plants

To investigate the biocontrol effectiveness of the antibiotic producing bacterium, Pseudomonas aureofaciens 63–28 against the phytopathogen Rhizoctonia solani AG-4 on Petri plates and in soybean roots, growth response and induction of PR-proteins were estimated after inoculation with P. aureofaciens 63–28 (P), with R. solani AG-4 (R), or with P. aureofaciens 63–28 + R. solani AG-4 (P + R). P. aureofaciens 63–28 showed strong antifungal activity against R. solani AG-4 pathogens in Petri plates. Treatment with P. aureofaciens 63–28 alone increased the emergence rate, shoot fresh weight, shoot dry weight and root fresh weight at 7 days after inoculation, when compared to R. solani AG-4; P + R treatment showed similar effects. Peroxidase (POD) and β-1,3-glucanase activity of P. aureofaciens 63–28 treated roots increased by 41.1 and 49.9%, respectively, compared to control roots. POD was 26% greater in P + R treated roots than R. solani treated roots. Two POD isozymes (59 and 27 kDa) were strongly induced in P + R treated roots. The apparent molecular weight of chitinase from treated roots, as determined through SDS-PAGE separation and comparison with standards, was about 29 kDa. Five β-1,3-glucanase isozymes (80, 70, 50, 46 and 19 kDa) were observed in all treatments. These results suggest that inoculation of soybean plants with P. aureofaciens 63–28 elevates plant growth inhibition by R. solani AG-4 and activates PR-proteins, potentially through induction of systemic resistance mechanisms.     

Bulb and Root Rot in Lily (Lilium longiflorum) and Onion (Allium cepa) in Israel

In the past 10 years, there has been a substantial increase in reports, from growers and extension personnel, on bulb and root rots in lily (Lilium longiflorum) in Israel. Rot in these plants, when grown as cut flowers, caused serious economic damage expressed in reduction in yield and quality. In lily, the fungal pathogens involved in the rot were characterized as binucleate Rhizoctonia AG‐A, Rhizoctonia solani, Pythium oligandrum, Fusarium proliferatum (white and purple isolates) and F. oxysporum, using morphological and molecular criteria. These fungi were the prevalent pathogens in diseased plants collected from commercial greenhouses. Pathogenicity trials were conducted on lily bulbs and onion seedlings under controlled conditions in a greenhouse to complete Koch's postulates. Disease symptoms on lily were most severe in treatments inoculated with binucleate Rhizoctonia AG‐A, P. oligandrum and F. proliferatum. Plant height was lower in the above treatments compared with the control plants. The least aggressive fungus was R. solani. In artificial inoculations of onion, seedling survival was significantly affected by all fungi. The most pathogenic fungus was F. proliferatum w and the least were isolates of F. oxysporum (II and III). All fungi were successfully re‐isolated from the inoculated plants.     

Characterization of Rhizoctonia solani associated with Sore Shin and Leaf Spot Symptoms on Tobacco in Zimbabwe

The relationship between Rhizoctonia solani isolates derived from leaf spot and sore shin symptoms on tobacco was characterized by determining nuclear number, hyphal diameter, anastomosis grouping (AG) and pathogenicity. The leaf spot isolates had significantly more nuclei per cell, and wider hyphae than the sore shin isolates. Anastomosis pairings with known tester strains confirmed the leaf spot isolates to be AG 3, and the sore shin isolates to be AG 4. Pathogenicity testing indicated that the leaf spot isolates were pathogenic to all parts of the plant, whereas the sore shin isolates tended to form lesions on stems only.