Effect of polyacetylenic acids from Prunella vulgaris on various plant pathogens

Aims: This study is aiming at characterizing antifungal substances from the methanol extract of Prunella vulgaris and at investigating those substances’ antifungal and antioomycete activities against various plant pathogens. Methods and Results: Two polyacetylenic acids were isolated from P. vulgaris as active principles and identified as octadeca‐9,11,13‐triynoic acid and trans‐octadec‐13‐ene‐9,11‐diynoic acid. These two compounds inhibited the growth of Magnaporthe oryzae, Rhizoctonia solani, Phytophthora infestans, Sclerotinia sclerotiorum, Fusarium oxysporum f. sp. raphani, and Phytophthora capsici. In addition, these two compounds and the wettable powder‐type formulation of an n‐hexane fraction of P. vulgaris significantly suppressed the development of rice blast, tomato late blight, wheat leaf rust, and red pepper anthracnose. Conclusions: These data show that the extract of P. vulgaris and two polyacetylenic acids possess antifungal and antioomycete activities against a broad spectrum of tested plant pathogens. Significance and Impact of the Study: This is the first report on the occurrence of octadeca‐9,11,13‐triynoic acid and trans‐octadec‐13‐ene‐9,11‐diynoic acid in P. vulgaris and their efficacy against plant diseases. The crude extract containing the two polyacetylenic acids can be used as a natural fungicide for the control of various plant diseases.     

Some fungal endophytes from vegetable crops and their anti-oomycete activities against tomato late blight

AIMS: To isolate endophytic fungi from vegetable plants and examine their in vivo anti-oomycete activity against Phytophthora infestans in tomato plants. METHODS AND RESULTS: Endophytic fungi were isolated from surface-sterilized plant tissues and anti-oomycete activity was measured by in vivo assay using tomato seedlings. Endophytic fungi showing potent anti-oomycete activity were identified by morphological characteristics and nuclear ribosomal ITS1-5.8S-ITS2 sequence analysis. A total of 152 isolates were obtained from 66 healthy tissue samples of cucumber, red pepper, tomato, pumpkin and Chinese cabbage and the fermentation broths of 23 isolates showed potent in vivo anti-oomycete activity against tomato late blight with control values over 90%. The Fusarium oxysporum strain EF119, which was isolated from roots of red pepper, showed the most potent disease control efficacy against tomato late blight. In dual-culture tests, it inhibited the growth of Pythium ultimum, P. infestans and Phytophthora capsici. CONCLUSIONS: Among endophytic fungi isolated from healthy tissues of vegetable plants, F. oxysporum EF119 showed the most potent in vivo anti-oomycete activity against tomato late blight and in vitro anti-oomycete activity against several oomycete pathogens. SIGNIFICANCE AND IMPACT OF THE STUDY: Endophytic fungi showing anti-oomycete activity in vitro and in vivo may be used as biocontrol agents particularly of tomato late blight.     

Runaway repeats force expansion of the Phytophthora infestans genome

Sequencing of the genome of the potato late blight pathogen Phytophthora infestans provides insight into genome structure and evolution within this genus of plant pathogenic oomycetes.     

The relationship between pathogenic fungal metabolities (fusaric and picolinic acid), endogenous ethylene evolution and the development of ethylene-like symptoms

Summary The effects of fusaric acid (5-n-butylpicolinic acid), picolinic acid (2-pyridine carboxylic acid), and picloram (4-amino-3, 5, 6-trichloropicolinic acid) on endogenous ethylene production by tomato cuttings and elongation growth of oat coleoptile sections were measured. Ethylene production by tomato cuttings was substantially stimulated by treatment with 1×10⁻³ and 1×10⁻⁵ M picoloram and to a lesser extent by 1×10⁻³ M fusaric acid; picolinic acid had little effect. The ethylene levels produced in response to fusaric acid are not high enough to account for the ethylene injury observed in Fusarium wilt. Fusaric acid inhibited oat coleoptile extension, picolinic acid had little effect, and picloram promoted growth.     

Activity of some aminoglycoside antibiotics against true fungi, Phytophthora and Pythium species

AIMS: To investigate the in vitro antifungal and antioomycete activities of some aminoglycosides against true fungi and Phytophthora and Pythium species and to evaluate the potential of the antibiotics against Phytophthora late blight on plants. METHODS AND RESULTS: Antifungal and antioomycete activities of aminoglycoside antibiotics (neomycin, paromomycin, ribostamycin and streptomycin) and a paromomycin-producing strain (Streptomyces sp. AMG-P1) against Phytophthora and Pythium species and 10 common fungi were measured in potato dextrose broth (PDB) and on seedlings in pots. Paromomycin was the most active against Phytophthora and Pythium species with a minimal inhibitory concentration of 1-10 microg ml(-1) in PDB, but displayed low to moderate activities towards other common fungi at the same concentration. Paromomycin also showed potent in vivo activity against red pepper and tomato late blight diseases with 80 and 99% control value, respectively, at 100 microg ml(-1). In addition, culture broth of Streptomyces sp. AMG-P1 as a paromomycin producer exhibited high in vivo activity against late blight at 500 microg freeze-dried weight per millilitre. CONCLUSIONS: Among tested aminoglycoside antibiotics, paromomycin was the most active against oomycetes both in vitro and in vivo. SIGNIFICANCE AND IMPACT OF THE STUDY: Data from this study show that aminoglycoside antibiotics have in vitro and in vivo activities against oomycetes, suggesting that Streptomyces sp. AMG-P1 may be used as a biocontrol agent against oomycete diseases.     

Antifungal activity of streptavidin C1 and C2 against pathogens causing Fusarium wilt

Fusarium wilt is caused by the soil-inhabiting fungus Fusarium oxysporum ff. spp. and is one of the most devastating plant diseases, resulting in losses and decreasing the quality and safety of agricultural crops. We recently reported the structures and biochemical properties of two biotin-binding proteins, streptavidin C1 and C2 (isolated from Streptomyces cinnamonensis strain KPP02129). In the present study, the potential of the biotin-binding proteins as antifungal agent for Fusarium wilt pathogens was investigated using recombinant streptavidin C1 and C2. The minimum inhibitory concentration of streptavidin C2 was found to be 16 µg ml^–1 for inhibiting the mycelial growth of F. oxysporum f.sp. cucumerinum and F. oxysporum f.sp. lycopersici, while that of streptavidin C1 was found to be 64 µg ml^–1. Compared with the nontreated control soil, the population density of F. oxysporum f.sp. lycopersici in the soil was reduced to 49·5% and 39·6% on treatment with streptavidin C1 (500 µg ml^–1) and C2 (500 µg ml^–1), respectively. A greenhouse experiment revealed that Fusarium wilt of tomato plants was completely inhibited on soil drenching using a 50-ml culture filtrate of the streptavidin-producing strain KPP02129.     

Mycorrhizal fungi induced activation of tomato defense system mitigates Fusarium wilt stress

The fungus Fusarium oxysporum f. sp. lycopersici (FOL) is known to cause vascular wilt on tomato almost over the world. Inoculation of FOL reduced plant growth and increased wilt of tomato. The following study examined the possible role of arbuscular mycorrhizal fungi (AMF) consortium comprising of Rhizophagus intraradices, Funneliformis mosseae and Claroideoglomus etunicatum against FOL in tomato and explored in an inducing plant systemic defense. AMF inoculation reduced the wilt disease within vascular tissue and in vivo production of fusaric acid was observed which may be responsible in reduced wilting. FOL had an antagonistic effect on AMF colonization, reduced the number of spores, arbuscules and vesicles. AMF also inhibited the damage induced by Fusarium wilt through increasing chlorophyll contents along with the activity of phosphate metabolising enzymes (acid and alkaline phosphatases). Moreover, tomato plants with mycorrhizal inoculation showed an increase in the level of antioxidant enzymes including glutathione reductase, catalase, and etc. with an ultimate influence on the elimination of reactive oxygen species. Moreover, rise in phosphatase along with antioxidant enzymatic systems and enhanced photosynthetic performance contributed to induced resistance against FOL in tomato.     

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.     

苏云金芽胞杆菌抗菌脂肽初步鉴定及抑菌活性测定

植物枯萎病是影响作物生长的重要因素，利用植物内生菌拮抗病原菌生长，从而降低其危害程度是目前研究的热点。本研究从健康的番茄植株中筛选分离得到一株对番茄枯萎病病原菌有较强拮抗作用的内生细菌B-R1,通过形态学、生理生化以及分子生物学检测分析，鉴定该菌株为苏云金芽胞杆菌(Bacillus thuringiensis)。为进一步探索该菌株的生防作用，首先将菌株发酵后离心，得到发酵上清液，采用盐酸沉淀法对其活性物质进行粗提，并检测其对大肠埃希菌(Escherichia coli)、金黄色葡萄球菌(Staphylococcus aureus)以及尖孢镰刀菌(Fusarium oxysporum)的抑菌活性。通过薄层色谱法(TLC)、傅里叶红外光谱(FI-TR)、高分辨液相色谱-质谱联用仪(LC-MS)分析并鉴定活性物质的结构。结果表明，苏云金芽胞杆菌对尖胞镰刀菌有良好的抑制作用，结构分析初步鉴定抗菌物质中含有丰原素(fengycin),属于脂肽类抗生素。     

In vitro kinetics and antifungal activity of various extracts of Terminalia chebula seeds against plant pathogenic fungi

The aim of the present study was to examine the efficacy of various seed extracts of Terminalia chebula as an antifungal potential against certain important plant pathogenic fungi. The organic extracts of methanol, ethyl acetate and chloroform at the used concentration of 1500 ppm/disc revealed remarkable antifungal effect as a fungal mycelial growth inhibitor against Fusarium oxysporum, Fusarium solani, Phytophthora capsici and Botrytis cinerea, in the range of 41.6–61.3%, along with MIC values ranging from 62.5 to 500 μg/ml. Also, the extracts had a strong detrimental effect on spore germination of all the tested plant pathogens along with concentration as well as time-dependent kinetic inhibition of B. cinerea. The results obtained from this study suggest that the natural products derived from Terminalia chebula could become an alternative to synthetic fungicides for controlling such important plant pathogenic fungi.     

Reaction of Certain Solanaceous and Asteraceous Plant Species to Inoculation with Phytophthora infestans in Cameroon

Experiments were conducted to detect potential hosts of Phytophthora infestans, causal agent of potato late blight among weeds occurring in Cameroon. Isolates of P. infestans isolated from garden huckleberry (Solanum scabrum), potato (S. tuberosum) and tomato (S. lycopersicon) were inoculated on detached leaves of 12 solanaceous and 14 asteraceous species collected from the potato agroecosystem in the western highlands of Cameroon. Isolates of P. infestans from huckleberry and potato infected the same host plants as well as gboma eggplant (S. macrocarpon) and two asteraceous weeds; Billy goatweed (Ageratum conyzoides) and Dichrocephala (Dichrocephala integrifolia). Inoculum from potato caused late blight symptoms on haemorrhage plant (Aspilia africana); while inoculum from tomato resulted in late blight on worowo (Solanecio biafrae). This is the first report of late blight infection on S. macrocarpon, A. conyzoides, Sol. biafrae and Asp. africana in Cameroon. The research results indicate that some asteraceous and solanaceous weeds may be alternative hosts of P. infestans in the potato agroecosystem.     

Growth inhibition of plant pathogenic fungi by hydroxy fatty acids

Hydroxy fatty acids are plant self-defense substances (Masui et al, Phytochemistry1989). Three types of hydroxy fatty acids: 10-hydroxystearic acid (HSA), 7S,10S-dihydroxy-8(E)-octadecenoic acid (DOD), and 12,13,17-trihydroxy-9(Z)-octadecenoic acid (THOA) were tested against the following plant pathogenic fungi: Erysiphe graminis f sp tritici (common disease name, wheat powdery mildew); Puccinia recondita (wheat leaf rust); Pseudocercosporella herpotrichoides (wheat foot rot); Septoria nodorum (wheat glume blotch); Pyricularia grisea (rice blast); Rhizoctonia solani (rice sheath blight); Phytophthora infestans (potato late blight); and Botrytis cinerea (cucumber botrytis). At a concentration of 200 ppm, both HSA and DOD showed no fungal disease control activity. However, THOA at the same concentration showed weak activity and provided disease control (percent) of the following plant pathogenic fungi: Erysiphe graminis 77%; Puccinia recondita 86%; Phytophthora infestans 56%; and Botrytis cinerea 63%. The position of the hydroxy groups on the fatty acids seems to play an important role in activity against specific fungi. Journal of Industrial Microbiology & Biotechnology (2000) 24, 275–276. Received 12 August 1999/ Accepted in revised form 06 January 2000     

Biotic and abiotic initiators for rishitin formation and accumulation in tomato

γ-Ray irradiation of pre-sowing seeds of tomato did not trigger the formation of the phytoalexin “rishitin” in either leaves or fruits of tomato plants through different growth seasons. Application of copper sulfate initiated rishitin formation in both leaves and fruits. Increasing of γ-ray dose was accompanied by decreasing rishitin accumulation in the presence of copper sulfate. Rishitin of tomato leaves was found to be reduced significantly, concomitant with increasing the disease incidence for late and early blight, andFusarium wilt disease, after applying γ-irradiation, in the case of biotic initiatorsPhytophthora infestans, Alternaria solani orFusarium oxysporum alone or together with the abiotic inducer copper sulfate. Shelf-extending γ-ray doses of 1.0, 1.5 and 2.0 kGy decreased rishitin amounts in tomato fruits treated with copper sulfate alone or infected withPhytophthora infestans. Also, the amount of formed rishitin was reduced by extending the storage period.     

Isolation,characterisation of major secondary metabolites of the Himalayan Trichoderma koningii and their antifungal activity

Ethyl acetate extracts of two strains of Trichoderma koningii were evaluated for antifungal activity against soilborne pathogenic fungi, Rhizoctonia solani, Sclerotium rolfsii, Macrophomina phaseolina and Fusarium oxysporum by poisoned food technique. Secondary metabolites, namely δ-decanolactone, 6-pentyl-α-pyranone and 6-(4-oxopentyl)-2H-pyran-2-one were isolated from T. koningii (T-8) extract while palmitic acid, 6-pentyl-α-pyranone and stigmasterol were isolated from T. koningii (T-11) extract. Secondary metabolites were identified by ¹H NMR, ¹³C NMR and mass spectroscopic methods. These metabolites were evaluated for antifungal activity against the test pathogens 6-Pentyl-α-pyranone and 6-(4-oxopentyl)-2H-pyran-2-one exhibited excellent antifungal activity against S. rolfsii. The antifungal activity though slightly less was comparable to that of hexaconazole, a commercial fungicide.     

Agrobacterium-Mediated Transformation of Tomato with rolB Gene Results in Enhancement of Fruit Quality and Foliar Resistance against Fungal Pathogens

Tomato (Solanum lycopersicum L.) is the second most important cultivated crop next to potato, worldwide. Tomato serves as an important source of antioxidants in human diet. Alternaria solani and Fusarium oxysporum cause early blight and vascular wilt of tomato, respectively, resulting in severe crop losses. The foremost objective of the present study was to generate transgenic tomato plants with rolB gene and evaluate its effect on plant morphology, nutritional contents, yield and resistance against fungal infection. Tomato cv. Rio Grande was transformed via Agrobacterium tumefaciens harbouring rolB gene of Agrobacterium rhizogenes. rolB. Biochemical analyses showed considerable improvement in nutritional quality of transgenic tomato fruits as indicated by 62% increase in lycopene content, 225% in ascorbic acid content, 58% in total phenolics and 26% in free radical scavenging activity. Furthermore, rolB gene significantly improved the defence response of leaves of transgenic plants against two pathogenic fungal strains A. solani and F. oxysporum. Contrarily, transformed plants exhibited altered morphology and reduced fruit yield. In conclusion, rolB gene from A. rhizogenes can be used to generate transgenic tomato with increased nutritional contents of fruits as well as improved foliar tolerance against fungal pathogens.     

Generation and characterization of reduced virulence Fusarium oxysporum f. sp. lycopersici mutants through plasmid-vector insertion

Pathogenicity-impaired mutants, B02 and H15, of Fusarium oxysporum f. sp. lycorpersici (FOL) were obtained using restriction enzyme-mediated integration. Disease severities of Fusarium wilt caused by these mutants were significantly reduced, and their disease development rates were correlated with their colonization rates in tomato vessels. Both B02 and H15 produced significantly smaller amounts of extracellular proteins as well as fusaric acid than the wild-type. Southern blot analyses suggested that B02 and H15 likely contain a single and three copies of transformation vector, respectively. These mutants may thus be useful in isolating genes involved in pathogenicity of FOL.     

Integrating cultural control methods for tomato late blight (Phytophthora infestans) in Uganda

Cultural control measures against tomato late blight (Phytophthora infestans) were evaluated in six field experiments over 3 years in Uganda. Each experiment included sanitation (removal of diseased plant tissues), fungicide (mancozeb) application, and an untreated control, as standard treatments. Late blight incidence and severity were greatly reduced by sanitation, without reducing the number of healthy leaves; however, tomato growth and production were adversely affected. Fungicide treated plants retained the highest numbers of flowers and attached fruits and gave the highest yields. Three cultural practices were evaluated in repeated experiments for their effectiveness in alleviating the adverse effects of sanitation. Tomatoes grown within plastic shelters early in the production cycle were taller, and had more healthy leaves than those grown late. The numbers of diseased leaves and disease severity were equally low in sanitation alone and plastic shelter/with sanitation treatments. Flower and fruit production were significantly higher when tomatoes were grown under early shelters with sanitation than with sanitation alone. Planting density was increased without significant effects on late blight and tomato growth and production. Intercropping tomato with soybean (Glycine max) or sesame (Sesamum indicum), with sanitation, limited late blight development, but taller intercrops suppressed tomato growth and production. Integrated treatments (combining plastic shelters, a sesame intercrop and high tomato planting density) were evaluated, with and without sanitation, against the fungicide mancozeb. The mean numbers of healthy leaves in the integrated treatments were not significantly less than with fungicide treatment. Late blight incidence and severity were higher in the integrated plots without than with sanitation. The numbers of flowers and attached fruits were not significantly less in integrated treatments than in fungicide treated plots, but tomato yield was highest with fungicide treatment.     

Time course study on accumulation of cell wall-bound phenolics and activities of defense enzymes in tomato roots in relation to <Emphasis Type="Italic">Fusarium</Emphasis> wilt

Major cell wall-bound phenolic compounds were detected and identified in roots of tomato at different stages of growth. Alkaline hydrolysis of the cell wall material of the root tissues yielded ferulic acid as the major bulk of the phenolic compounds. Other phenolic compounds identified were 4-hydroxybenzoic acid, vanillic acid, 4-hydroxybenzaldehyde, vanillin and 4-coumaric acid. All the six phenolic acids were higher in very early stage of plant growth. Ferulic acid, 4-hydroxybenzoic acid and 4-coumaric acid exhibited a decreasing trend up to 60 days and then the content of these phenolic acids increased somewhat steadily towards the later stage of growth. Total phenolics, phenylalanine ammonia-lyase (PAL) activity and peroxidase (POD) activity were in tandem match with the occurrence pattern of the phenolic acids. Ferulic acid showed highest antifungal activity against tomato wilt pathogen Fusarium oxysporum f. sp. lycopersici. The results of this study may be interpreted to seek an explanation for high susceptibility of tomato plants at flowering stage to Fusarium wilt. It may also be concluded that greater amounts of ferulic acid in combination with other phenolics and higher level of PAL and POD activities after 60 days of growth may have a role in imparting resistance against Fusarium wilt at a late stage of plant growth.