Fungicidal Activity of Volatiles from Selected Cruciferous Plants against Resting Propagules of Soil-borne Fungal Pathogens

The efficacy of volatiles evolved from tissues of nine cruciferous plants against resting propagules of Fusarium oxysporum var radicis f. sp. lycopersici, Sclerotium cepivorum, and Sclerotinia sclerotiorum was tested. The cruciferous plants released biocidal compounds, mainly isothiocyanates, produced during the enzymatic degradation of glucosinolates present in the plant cells. Among the plants investigated, the highest fungicidal activity and also the highest concentration of isothiocyanates were found in Brassica juncea. The resting propagules of tested fungi differed significantly in their sensitivity towards volatiles released from plant tissues.     

The effect of species of Allium on soil bacteria in relation to germination of sclerotia of Sclerotium cepivorum Berk

Extracts made from Allium species were shown to exhibit marked antibiotic properties in a variety of tests. However, no evidence was obtained to suggest that intact Allium seedlings exude antibiotic compounds in sufficient quantity to cause inhibition of bacteria in the soil or in assay tests in the laboratory. Germination of sclerotia of Sclerotium cepivorum was induced by Allium extracts which were too dilute to cause antibiotic responses in laboratory tests using a number of bacteria. Synthetic methyl-methanethiolsulphinate had little effect on germination of sclerotia. The specific reversal, by Allium species and their extracts, of the mycostatic effect of unsterile soil on sclerotia of S. cepivorum does not appear to be due to antibiotic effects.     

Germination of sclerotia of Sclerotium cepivorum Berk. under aseptic conditions

The response of sclerotia of Sclerotium cepivorum to a number of plant extracts and nutrient solutions was tested, aseptically, on agars, silica gels, filter paper discs, and on columns of soil. There was no specific response of sclerotia to extracts of Allium species in any of these experiments, whereas in unsterile soil consistently high levels of germination were obtained only with Allium extracts. It was shown that sclerotia are subject to the mycostatic influence of unsterile soil. With S. cepivorum soil mycostasis appears to be specifically reversed by Allium species or by water extracts made from them.     

Endophytic Bacteria Induce Growth Promotion and Wilt Disease Suppression in Oilseed Rape and Tomato

To determine whether bacteria isolated from within plant tissue can have plant growth-promotion potential and provide biological control against soilborne diseases, seeds and young plants of oilseed rape (Brassica napus L. cv. Casino) and tomato (Lycopersicon lycopersicum L. cv. Dansk export) were inoculated with individual bacterial isolates or mixtures of bacteria that originated from symptomless oilseed rape, wild and cultivated. They were isolated after surface sterilization of living roots and stems. The effects of these isolates on plant growth and soilborne diseases for oilseed rape and tomato were evaluated in greenhouse experiments. We found isolates that not only significantly improved seed germination, seedling length, and plant growth of oilseed rape and tomato but also, when used for seed treatment, significantly reduced disease symptoms caused by their vascular wilt pathogens Verticillium dahliae Kleb and Fusarium oxysporum f. sp. lycopersici (Sacc.), respectively.     

Factors affecting the survival of sclerotia of Sclerotium cepivorum in the Fraser Valley of British Columbia

Sclerotia of Sclerotium cepivorum buried in muck soil in the Fraser Valley decayed with time. The rate of decay of sclerotia was influenced by local environmental conditions. A mixture of soil with sclerotia increased their survival but there was no difference in the rates of decay in three different soils. The decay was greatest during winter when Fraser Valley fields are often flooded. Sclerotial decay was also affected by pretreatment of the sclerotia. Dried sclerotia decayed significantly (P < 0.05) faster than sclerotia which had not been dried, a phenomenon which is apparently due to changes in micro-organisms on the sclerotia. Dried sclerotia which had been incubated in moist soil had fewer bacteria and more fungi than sclerotia which had been incubated in soil without being dried. The increase in fungi on the dried sclerotia was due to a dramatic increase in Trichoderma spp.     

Biological control of white rot of onion

Summary Interactions of 123 isolates of fungi, 17 of bacteria and 22 of actinomycetes, respectively, withSclerotium cepivorum were studied in agar culture. They were grouped into 4 different types of reactions. Amongst themTrichoderma viride, Fusarium graminearum, Coniothyrium minitans andGliocladium roseum inhibited the growth ofS. cepivorum and later grew over its colony.T. viride showed a characteristic coiling around the hyphae ofS. cepivorum. T. viride andF. graminearum prevented the development of sclerotia.C. minitans was found to parasitize the sclerotia ofS. cepivorum and produced its pycnidia within them.Aleurisma carnis, Cladosporium elatum, Penicillium expansum, P. nigricans, P. notatum, P. piscarium, P. puberulum, P. rolfsii, P. urticae, P. variabile, Tilachlidium humicola andHelminthosporium sp. inhibited the growth ofS. cepivorum at a distance. Eleven isolates of bacteria and 3 ofStreptomyces sp. showed pronounced antagonistic properties againstS. cepivorum.Experiments were carried out to study the effects on white rot development in soil of organisms selected from agar plate tests. None of the antagonistic micro organisms had any deleterious effects on onion growth. Of the organisms testedP. nigricans gave the best results in controlling white rot infection.This work was carried out at the Department of Botany, The University, Birmingham, England. I wish to thank Prof.C. J. Hickman for his invaluable advice and encouragement.     

Effect of Brassica Biofumigant Amendments on Different Stages of the Life Cycle of Phytophthora cinnamomi

The oomycete plant pathogen Phytophthora cinnamomi causes a highly destructive root rot that affects numerous hosts. Integrated management strategies are needed to control P. cinnamomi in seminatural oak rangelands. We tested how biofumigation affects crucial stages of the pathogen's life cycle in vitro, in infested soils under laboratory conditions and in planta. Different genotypes of three potential biofumigant plant species (Brassica carinata, Brassica juncea, Brassica napus) were collected at different phenological stages, analysed for their glucosinolate contents, and subsequently tested. The most effective genotypes against mycelial growth and sporangial production were further tested on the viability of chlamydospores in artificially infested natural soils and in planta on Lupinus luteus, a host highly susceptible to P.cinnamomi. Brassica carinata and B. juncea genotypes inhibited mycelial growth, decreased sporangial production, and effectively inhibited the viability of chlamydospores in soil, but only B. carinata decreased disease symptoms in plants. Effective genotypes of Brassica had high levels of the glucosinolate sinigrin. Biofumigation with Brassica plants rich in sinigrin has potential to be a suitable tool for control of oak root disease caused by P. cinnamomi in Spanish oak rangeland ecosystems.     

Response of different forms of propagules of Rhizoctonia solani AG2-1 (ZG5) exposed to the volatiles produced in soil amended with green manures

The sensitivity of different forms of propagules of Rhizoctonia solani anastomosis group (AG)2‐1/zymogram group (ZG)5 to volatile compounds produced in soil amended with green manure will influence the efficacy of green manures used to manage the disease. In laboratory experiments, we determined the impact of volatiles arising from residues of five species of Brassicaceae, and Avena sativa (oat), a non‐Brassicaceae species, and volatiles of pure allyl isothiocyanate (AITC) or 2‐phenylethyl isothiocyanate (2‐PEITC) in either their soluble or vapour phase on the hyphal growth of R. solani arising from different propagules. The brassicaceous species were Brassica napus var. Karoo, B. napus B1, B. napus B2, B. nigra and Diplotaxis tenuifolia (a brassicaceous weed). Colony growth and hyphal density on water agar were measured up to 7 days. The amendment of a sandy soil with green manures at a high (100 g kg^?1, 10%) concentration generally suppressed the growth of the pathogen, but at a low (10 g kg^?1, 1%) concentration, the amendment had little effect on the radial fungal growth of the pathogen but increased the density of hyphae through more branching. The inhibition by volatiles from the residues of Brassicaceae species at 10% concentration was stronger (82–86%) than that by volatiles from oat (64%) amendment at the same rate. Hyphae arising from sclerotia and precolonised ryegrass seed were less sensitive than hyphae growing out of potato dextrose agar plugs. This indicates that thick‐pigmented cell walls may have protected the fungus from these unfavourable conditions. Pure AITC and 2‐PEITC in the range of 0.5–2.0 mM inhibited the growth of R. solani from all forms of propagules, but hyphae originating from agar plugs were the most vulnerable compared with the two others. Thus, hyphae arising from the medulla of the sclerotia may be relatively tolerant to volatile compounds emanating from decomposing Brassica green manure amendments in the field and in vitro inhibition of the vegetative growth of the pathogen may not reflect its response to the amendments in the field.     

Development of a Loop‐Mediated Isothermal Amplification Assay to Detect Fusarium oxysporum

We report the development of a loop‐mediated isothermal amplification (LAMP) assay targeting the CYP51C element for visual detection of F. oxysporum which caused Fusarium wilt in soybean. The CYP51C‐LAMP assay efficiently amplified the target gene in 60 min at 62°C. And specificity was evaluated against F. oxysporum, Fusarium spp. and other fungal species. The detection limit of the CYP51C‐specific LAMP assay for F. oxysporum was four conidia per gram soil. The assay also detected F. oxysporum from inoculated soybean tissues and residues. These results suggest that this CYP51C‐LAMP assay can be used to detect residues on plants in the field.     

Secondary metabolites produced by a root‐inhabiting sterile fungus antagonistic towards pathogenic fungi

Aims: A sterile red fungus (SRF) isolated from cortices of roots of both wheat (Triticum aestivum cv. Gamenya) and ryegrass (Lolium rigidum cv. Wimmera) was found to protect the hosts from phytopathogens and promote plant growth. In this work, the major secondary metabolites produced by this SRF were analysed, and their antibiotic and plant‐growth‐promoting activities investigated. Methods and Results: Two main compounds, veratryl alcohol (VA) and 4‐(hydroxymethyl)‐quinoline, were isolated from the culture filtrate of the fungus. In antifungal assays, VA inhibited the growth of Sclerotinia sclerotiorum and Pythium irregulare even at low amounts, while high concentrations (>100 μg per plug) of 4‐(hydroxymethyl)‐quinoline were needed. Both metabolites revealed weak inhibition of Rhizoctonia solani. Furthermore, both compounds showed a growth promotion activity on canola (Brassica napus) seedlings used as bioassays. Conclusions: Isolation and characterization of the main secondary metabolites from culture filtrates of a root‐inhabiting sterile fungus are reported. The biological assays indicate that these secondary metabolites may have a role in both plant growth regulation and antifungal activity. Significance and Impact of the Study: This study provides a better understanding of the metabolism of a cortical fungus that may have a useful role in the biological suppression of root‐infecting soil‐borne plant pathogens.     

A proteomics approach to study synergistic and antagonistic interactions of the fungal–bacterial consortium Fusarium oxysporum wild‐type MSA 35

Fusarium oxysporum is an important plant pathogen that causes severe damage of many economically important crop species. Various microorganisms have been shown to inhibit this soil‐borne plant pathogen, including non‐pathogenic F. oxysporum strains. In this study, F. oxysporum wild‐type (WT) MSA 35, a biocontrol multispecies consortium that consists of a fungus and numerous rhizobacteria mainly belonging to γ‐proteobacteria, was analyzed by two complementary metaproteomic approaches (2‐DE combined with MALDI‐Tof/Tof MS and 1‐D PAGE combined with LC‐ESI‐MS/MS) to identify fungal or bacterial factors potentially involved in antagonistic or synergistic interactions between the consortium members. Moreover, the proteome profiles of F. oxysporum WT MSA 35 and its cured counter‐part CU MSA 35 (WT treated with antibiotics) were compared with unravel the bacterial impact on consortium functioning. Our study presents the first proteome mapping of an antagonistic F. oxysporum strain and proposes candidate proteins that might play an important role for the biocontrol activity and the close interrelationship between the fungus and its bacterial partners.     

Bacterial siderophores promote plant growth: Screening of catechol and hydroxamate siderophores

The aim of the study was to determine the quality and quantity of siderophores produced by bacteria isolated from plants' roots. The second aim was to determine the effect of siderophores on plants growth (Festuca rubra L. and Brassica napus L.). The study was carried out using bacteria isolated from roots of: Arabidopsis thaliana L., F. rubra, and Agrostis capillaris L., growing on the heavy metals contaminated area. The chrome azurol sulfonate (CAS) test, Arnow's test for catechol siderophores, and Csaksy's test for hydroxamate siderophores were performed. Among the bacteria, 42 isolates (39%) had a positive result in the CAS. Endophytic bacteria were mostly producing the catechol siderophores. It was found that F. rubra is the plant which is linked with the highest number of siderophores producing bacteria. The highest concentration of siderophores was noted for ectorhizospheric bacteria associated with A. thaliana, hyperaccumulating plant. It was found that hydroxamate siderophores are mainly produced by ectorhizosphere and rhizoplane bacteria. The siderophores producing bacteria reduced the toxicity of metals and improved the phytoremediation. Siderophores treatment increased the growth of plants in the biological assay, growing on two different soils: one highly contaminated with heavy metals and the second strongly alkaline soil.     

Induced tolerance of Sclerotium cepivorum to antibiotics in the presence of onion exudates

The onion white rot pathogen Sclerotium cepivorum was cultured on agar media containing 2% malt extract and one of the antifungal antibiotics, endomycin, griseofulvin, venturicidin and cycloheximide at concentrations that reduced but did not prevent growth of mycelium. When onion seeds or agar discs impregnated with diffusates from onion bulbs were placed on the antibiotic media, radial growth of the fungus was greatly increased, and there was a profuse development of aerial mycelium. Gaseous diffusates from onion tissue and from impregnated agar discs were also effective. On the antibiotic media, tomato, cabbage and radish seeds did not stimulate the growth of S. cepivorum and the onion exudates did not stimulate the growth of four other fungi. This and other evidence is considered to show that the stimulation of growth of S. cepivorum was not caused by any direct effect on the antibiotics but by a tolerance of the fungus to them, which was specifically induced by an exudate from its host plant. The phenomenon may be related to the reported reversal by onion extracts of the inhibitory effects of soil mycostasis on germination of sclerotia of the fungus.     

Increased plant growth and copper uptake of host and non-host plants by metal-resistant and plant growth-promoting endophytic bacteria

The effects of inoculation with two metal-resistant and plant growth-promoting endophytic bacteria (Burkholderia sp. GL12 and Bacillus megaterium JL35) were evaluated on the plant growth and Cu uptake in their host Elsholtzia splendens and non-host Brassica napus plants grown in natural Cu-contaminated soil. The two strains showed a high level of ACC deaminase activities. In pot experiments, inoculation with strain GL12 significantly increased root and above-ground tissue dry weights of both plants, consequently increasing the total Cu uptake of E. splendens and Brassica napus by 132% and 48.2% respectively. Inoculation with strain JL35 was found to significantly increase not only the biomass of B. napus, consequently increasing the total Cu uptake of B. napus by 31.3%, but Cu concentration of E. splendens for above-ground tissues by 318% and roots by 69.7%, consequently increasing the total Cu uptake of E. splendens by 223%. The two strains could colonize the rhizosphere soils and root interiors of both plants. Notably, strain JL35 could colonize the shoot tissues and significantly increase the translocation factors and bioaccumulation factors of E. splendens. These results suggested that Burkholderia sp. GL12 and B. megaterium JL35 were valuable bacterial resource which had the potential in improving the efficiency of Cu phytoextraction by E. splendens and B. napus in a natural Cu-contaminated soil.     

Screening of oilseed rape and other brassicaceous genotypes for susceptibility to Ceutorhynchus pallidactylus (Mrsh.)

Production of oilseed rape, Brassica napus L., is affected by various insect pests. The cabbage stem weevil, Ceutorhynchus pallidactylus (Mrsh.) (Col.: Curculionidae), is one of the most damaging pests in Northern and Central Europe that requires regular control measures. Host plant resistance is a key factor in integrated pest management systems. To evaluate a large number of genotypes for their susceptibility to infestation by C. pallidactylus, new screening techniques were developed for testing both, the amount of feeding and the number of eggs deposited by adult C. pallidactylus on accessions of Brassicaceae under controlled conditions. In no‐choice screening tests, the leaf area consumed by adult cabbage stem weevil was quantified on a wide spectrum of 107 brassicaceous genotypes (B. napus, Brassica rapa L. and Brassica oleracea L. cultivars, breeding lines, resynthesized rapeseed lines and wild Brassicaceae). In comparison to feeding on the standard cultivar ‘Express’, the average leaf area consumed by C. pallidactylus on nine oilseed rape cultivars, four resynthesized rapeseed lines and five other accessions [B. oleracea, Camelina alyssum (Mill.) and Lunaria annua L.] was significantly reduced by 44–90%. In dual‐choice screening tests for the evaluation of oviposition preferences on 42 genotypes, female C. pallidactylus laid significantly fewer eggs into plants of two oilseed rape cultivars, five resynthesized rapeseeds and three accessions of B. oleracea and Brassica fruticulosa Cyrillo, respectively, than into plants of the standard cv ‘Express’. Results of both laboratory screening tests were confirmed by results of additional field testing.     

Reproductive Development in Oilseed Rape (Brassica napus cv. Bienvenu)

The reproductive development of oilseed rape (Brassica napusL. cv. Bienvenu) was studied using light and scanning electronmicroscopy. By using the two techniques on comparable samples,internal events such as microspore development were relatedto the morphology of the developing floral parts, and this inturn was linked to the growth stage of the plant. Reproductive development, oilseed rape, Brassica napus, growth stage     

Antagonistic Effect of Fusarium oxysporum on Heterodera cruciferae

This study was conducted to investigate the effects of Fusarium oxysporum Schlecht. (isolate no. B6) on Heterodera cruciferae Franklin and its pathogenicity on cabbage plants. Fusarium oxysporum (B6) was isolated from cysts extracted from cabbage (Brassica olareacae L.) fields in Samsun (north part of Turkey). Healthy cysts obtained from mass culture in a growing chamber were placed on fungal colonies already formed in Petri dishes. The highest infection rate was observed 10 days after inoculation and reached 72%. Fusarium oxysporum also had the ability to penetrate through cysts wall. The effects of spore suspensions of Fusarium on H. cruciferae in cabbage plants were tested in pot experiments in growth chambers. Cabbages cyst nematode (CCN) eggs were first incubated in a fungal spore suspension (10⁸–10⁹ spores/ml) for 5 min and then introduced into pots containing sterilized soil and 3‐weeks‐old healthy cabbage plants. A positive effect was observed on plant height, fresh weight, fresh root weight, length and leaf area. Nematode numbers in the root system decreased within 2 months after artificial inoculations with Fusarium‐infected cysts. This suggests a negative, antagonistic effect between F. oxysporum and H. cruciferae.     

Specific truncations of an acetolactate synthase gene from Brassica napus efficiently complement ilvB/ilvG mutants of Salmonella typhimurium

Summary The expression of an acetolactate synthase (ALS) gene isolated from the cruciferous plant Brassica napus was investigated in Salmonella typhimurium. Using an expression plasmid containing the highly active trc (trp-lac) promoter, several plant ALS constructs were made containing successive in-frame truncations from the 5 end of the coding region. Functional complementation by these plant ALS constructs of a S. typhimurium mutant devoid of ALS enzymic activity was assayed on minimal medium. Truncations which eliminated a large portion of the transit peptide coding sequence proved to act as efficient ALS genes in the bacterial host. Truncations close to the putative processing site of the plant protein were inactive in the complementation test. A full length copy of the gene, including the entire transit peptide coding region, was also inactive. The efficiency of the complementation, estimated by comparison to the growth rate of wild-type S. typhimurium, was found to correlate with levels of ALS activity in the transformed bacteria. Specific mutations, known to produce herbicide resistance in plants, were introduced into the truncated ALS coding sequence by site-directed mutagenesis. When expressed in bacteria these constructs conferred a herbicide resistance phenotype on the host. The potential of this system for mutagenesis and enzymological studies of plant proteins is discussed.     

Effect of Simulated Soil Solarization and Organic Amendments on Fusarium Wilt of Rocket and Basil Under Controlled Conditions

Pot trials were carried out under controlled conditions to evaluate the effectiveness against Fusarium wilt of rocket (Fusarium oxysporum f.sp. conglutinans) and basil (F. oxysporum f.sp. basilici) of soil amendments based on a patented formulation of Brassica carinata defatted seed meal and compost, combined or not with a simulation of soil solarization. The soil solarization treatment was carried out in a growth chamber by heating the soil for 7 and 14 days at optimal (55–52°C for 6 h, 50–48°C for 8 h and 47–45°C for 10 h/day) and sub‐optimal (50–48°C for 6 h, 45–43°C for 8 h and 40–38°C for 10 h/day) temperatures similar to those observed in summer in solarized soil in greenhouses in Northern Italy. Two subsequent cycles of plant cultivation were carried out in the same soil. Even at sub‐optimal temperature regimes, 7 days of thermal treatment provided very valuable results in terms of disease control on both rocket and basil. In general, the thermal treatment was more effective against F. oxysporum f.sp. basilici than against F. oxysporum f.sp. conglutinans. Control of Fusarium wilt of rocket is improved with 14 days of thermal treatment. The combination of organic amendments with a short period of soil solarization (7 or 14 days), although not providing any improvement to the level of disease management, did significantly increase biomass and positively affected yield.     

Effects of volatile products of Allium species and their extracts on germination of sclerotia of Sclerotiutn cepivorum Berk.

It was shown that sclerotia of Sclerotium cepivorum, normally subject to the mycostatic influence of unsterile soil, germinated in response to a volatile principle evolved from growing onion and leek seedlings. Air, which was bubbled through diluted onion and garlic juices, contained vapours which stimulated sclerotia! germination. The vapours evolved at room temperature by garlic juice were collected as a condensate which also induced germination. Stimulatory activity was found in both distillates and distillands of garlic as well as in air bubbled through such a distillate.