Biological control of fusarium crown and root rot of tomato with antagonistic bacteria and integrated control when combined with the fungicide carbendazim

Two bacterial isolates, Bacillus megaterium (c96) and Burkholderia cepacia (c91), demonstrated to be antagonistic against Fusarium oxysporum f.sp. radicis-lycopersici , the causal organism of fusarium crown and root rot of tomato, were evaluated as biocontrol agents alone and when integrated with the fungicide carbendazim. In an initial screening, these isolates reduced disease incidence by 75 and 88%, respectively. In vitro , both biocontrol agents were highly tolerant to the fungicide carbendazim, commonly used to control fusarium diseases. Carbendazim reduced disease symptoms by over 50% when used at > 50  µ g mL⁻¹, but had little effect at lower concentrations. Combination of the bacterial isolates and carbendazim gave significant ( P  ≤ 0·05) control of the disease when plants were artificially inoculated with the pathogen. Application of carbendazim at a low concentration (1  µ g mL⁻¹) in combination with B. cepacia c91 reduced disease symptoms by 46%, compared with a reduction of 20% obtained with the bacterium alone and no control with the chemical treatment alone. A combination of B. megaterium c96 with an increased application rate of 10  µ g mL⁻¹ carbendazim significantly reduced disease symptoms by 84% compared with inoculated controls and by 77% compared with carbendazim treatment alone. In this experiment, the integrated treatment also slightly outperformed application of 100  µ g mL⁻¹ carbendazim, and bacteria applied without fungicide also provided good disease control.     

Antifungal activity of bergenin, a constituent of Flueggea microcarpa

The antifungal activity of bergenin against some plant pathogenic fungi, namely, Alternaria alternata , A. brassicae , A. carthami , Fusarium udum , F. oxysporum f.sp. ciceri , Curvularia lunata and Erysiphe pisi , was studied. Bergenin as its monosodium salt was effective against all the fungi and the effective dose for complete inhibition of spore germination varied from 15 μg mL⁻¹ for F. udum to 125 μg mL⁻¹ for E. pisi . Experiments on the effect of bergenin on powdery mildew development under glasshouse conditions revealed that it can control powdery mildew of pea at 2000 μg mL⁻¹ by postinoculation treatment, the results being comparable with those of carbendazim (1000 μg mL⁻¹) and wettable sulfur (2000 μg mL⁻¹). It affected hyphal elongation and the number of primary and secondary branches.     

Management of late leaf spot of groundnut (Arachis hypogaea) with chlorothalonil-tolerant isolates of Pseudomonas aeruginosa

Fifteen groundnut-associated bacterial isolates that inhibited by > 90% the in vitro conidial germination of Phaeoisariopsis personata , causal agent of late leaf spot disease of groundnut, were applied as a prophylactic spray (10⁸ cfu mL⁻¹) and tested for control of the disease in the glasshouse. Two groundnut seed-associated bacterial isolates, GSE 18 and GSE 19, identified as Pseudomonas aeruginosa , reduced the lesion frequency (LF) by up to 70%. A 90-day-old peat-based formulation of P. aeruginosa GSE 18 reduced LF measured 15 days postinoculation by up to 60%. Both P. aeruginosa GSE 18 and GSE 19 were tolerant to chlorothalonil (Kavach®) up to 2000  µ g mL⁻¹ in LB broth. In glasshouse trials, GSE 18 and GSE 19 tested in combination with reduced concentrations of chlorothalonil were highly efficient in management of the disease. The disease was completely controlled by chlorothalonil (> 250  µ g mL⁻¹), and in the presence of GSE 18 or GSE 19, 100  µ g mL⁻¹ chlorothalonil was equally effective. Application of rifamycin-resistant mutants of GSE 18 or GSE 19 together with chlorothalonil significantly increased the survival of these isolates in the groundnut phylloplane. In the field, a combination of GSE 18 and 500  µ g mL⁻¹ chlorothalonil reduced disease severity comparable to 2000  µ g mL⁻¹ chlorothalonil alone. Use of chlorothalonil-tolerant pseudomonads together with a quarter concentration of the recommended field dose of chlorothalonil doubled pod yield compared with the untreated unsprayed control.     

Occurrence of Corynespora cassiicola isolates resistant to boscalid on cucumber in Ibaraki Prefecture, Japan

A total of 651 isolates of cucumber corynespora leaf spot fungus ( Corynespora cassiicola ) collected from cucumber in Japan, either with (438 isolates) or without (213 isolates) a prior history of boscalid use, were tested for their sensitivity to boscalid by using a mycelial growth inhibition method on YBA agar medium. Additionally, seven isolates of C. cassiicola obtained from tomato, soybean, eggplant (aubergine) and cowpea in different locations in Japan were tested before boscalid registration. Minimum inhibitory concentration (MIC) and 50% effective concentration (EC₅₀) values for 220 isolates from crops without a prior history of boscalid use ranged from 0·5 to 7·5 μg mL⁻¹ and from 0·04 to 0·59 μg mL⁻¹, respectively. Two hundred and fourteen out of 438 isolates collected from ten cucumber greenhouses in Ibaraki Prefecture, Japan, which received boscalid spray applications showed boscalid resistance, with MIC values higher than 30 μg mL⁻¹. Moreover, resistant isolates were divided into two groups: a moderately resistant (MR) group consisting of 189 isolates with EC₅₀ values ranging from 1·1 to 6·3 μg mL⁻¹, and a very highly resistant (VHR) group consisting of 25 isolates with EC₅₀ values higher than 24·8 μg mL⁻¹. MR isolates were detected from all ten greenhouses, but VHR isolates were detected from only three. As a result of fungus inoculation tests which used potted cucumber plants, control failures of boscalid were observed against resistant isolates. Efficacy of boscalid was remarkably low against VHR isolates in particular. This is the first known report on boscalid resistance in Japan.     

Interaction of a bioherbicide and glyphosate for controlling hemp sesbania in glyphosate-resistant soybean

The bioherbicidal fungus, Colletotrichum truncatum (Schwein.) Andrus & Moore, was tested at different inoculum concentrations alone and in combination with, prior to or following treatment with different rates of glyphosate ( N -[phosphonomethyl]glycine) (Roundup Ultra) for the control of hemp sesbania ( Sesbania exaltata [Raf.] Rydb. ex A.W. Hill) in Roundup Ready soybean field plots. Colletotrichum truncatum and glyphosate were applied in all pair-wise combinations of 0, 1.25, 2.5, 5.0, and 10.0 × 10⁶ spores mL⁻¹ (i.e. 3.125, 6.25, 12.5, and 25 × 10¹¹ spores ha⁻¹), and 0.15, 0.30, 0.60, and 1.2 kg ha⁻¹, respectively. Weed control and disease incidence were enhanced at the two lowest fungal and herbicidal rates when the fungal spores were applied after glyphosate treatment. The application of the fungus in combination with or prior to glyphosate application at 0.30 kg ha⁻¹ resulted in reduced disease incidence and weed control regardless of the inoculum's concentration. At the highest glyphosate rates, the weeds were controlled by the herbicide alone. These results suggest that it might be possible to utilize additive or synergistic herbicide and pathogen interactions to enhance hemp sesbania control.     

Antifungal vapour-phase activity of allyl-isothiocyanate against Penicillium expansum on pears

The fungitoxicity of allyl-isothiocyanate (AITC) vapour against Penicillium expansum , the causal agent of blue mould on pears (cvs Conference and Kaiser), was evaluated. The best control of blue mould was obtained by exposing fruits for 24 h in a 5 mg L⁻¹ AITC-enriched atmosphere, the extent of control depending on the inoculum density. Lesion diameter was inversely related to AITC concentration. In treated fruits the percentage of infected wounds increased with conidial concentration, with fewer than 20% affected at 1 × 10³ conidia mL⁻¹ to almost 80% at 1 × 10⁶ conidia mL⁻¹. In comparison, >98% of wounds were infected in untreated fruits irrespective of conidial concentration. AITC treatments were effective up to 24 h after inoculation for Conference and 48 h for Kaiser. AITC treatments also controlled a thiabendazole-resistant strain of P. expansum , reducing the incidence of blue mould by 90% in both cultivars. The use of AITC produced from pure sinigrin or from Brassica juncea defatted meal may be an economically viable alternative to synthetic fungicides against P. expansum .     

Sensitivity of Sclerotinia homoeocarpa to demethylation-inhibiting fungicides in Ontario, Canada, after a decade of use

In late 2003, nine populations of Sclerotinia homoeocarpa in Ontario Canada (seven of which had been previously sampled in early 1994, prior to the registration of sterol demethylation-inhibiting (DMI) fungicides for turf disease control in Canada) were sampled and tested for sensitivity to propiconazole. Four of the nine populations had not been treated with DMI fungicides during the intervening years, and isolates from these locations were sensitive to propiconazole (geometric mean EC₅₀ values of 0·005–0·012 µ g mL⁻¹, compared with 0·005–0·008 µ g mL⁻¹ for the original 1994 populations). Among the five populations from 2003 that had been exposed to DMI fungicides, mean EC₅₀ values were significantly greater, ranging from 0·020 to 0·048 µ g mL⁻¹. A significant correlation of determination was found between estimated number of fungicide applications and log EC₅₀ ( R ² = 0·832, P  = 0·0001), and the equation predicted that 42·3 applications of propiconazole would be needed to bring a sensitive population (EC₅₀ < 0·01  µ g mL⁻¹) to a resistant level (EC₅₀ > 0·10  µ g mL⁻¹). Fungicide sensitivity vs. duration of fungicide efficacy was also tested, and it was found that isolates with decreased sensitivity were able to more quickly overcome the inhibitory effects of fungicide application, reducing the duration of control from 3 weeks to 2 weeks.     

Effect of 2,6-dichloroisonicotinic acid, its formulation materials and benzothiadiazole on systemic resistance to alternaria leaf spot in cotton

A wettable powder (WP) formulation providing 5–25 μg mL⁻¹ of 2,6-dichloroisonicotinic acid (INA) and 15–75 μg mL⁻¹ of WP applied to cotton cotyledons significantly increased the resistance of the next two leaves to challenge inoculation by Alternaria macrospora . The wettable powder alone at 15–75 μg mL⁻¹ had a lesser effect. A wettable granule (WG) formulation supplying 35 μg mL⁻¹ of benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH) and 35 μg mL⁻¹ of WG, applied as a cotyledonary treatment, significantly reduced the formation of lesions on the subsequent two leaves when challenged with A. macrospora . The WG control had no effect. Each treatment except for the WG control also raised the activities of β-1,3-glucanase in unchallenged leaf and stem tissue. Each of the components of the wettable powder without INA applied to cotyledons raised enzyme activities in the next leaves. Individual components, as suspensions of silicic acid and kaolin and solutions of the detergent Attisol II, the wetting agent Ultravon W300 and pure INA, applied to cotyledons increased the resistance of the next leaves to A. macrospora . The responsiveness of cotton to BTH and to each of the components of formulated INA is discussed in relation to knowledge of the effects of BTH and INA on other plants and to possible ways in which the other components of the wettable powder may affect the process of signalling for systemic resistance to disease.     

Control of soilborne clubroot disease of cruciferous plants by epoxydon from Phoma glomerata

Culture broth from an isolate of Phoma glomerata (no. 324, = JCM 9972) from the leaves of Viola sp., controlled the soilborne pathogen Plasmodiophora brassicae which causes clubroot disease of cruciferous plants. This effect was caused by epoxydon (5-hydroxy-3-(hydroxymethyl)-7-oxabicyclo[4.1.0]hept-3-en-2-one). Although this substance was known to have antitumour activity, phytotoxicity and antiauxin activity, no plant disease reduction had been reported previously. Epoxydon possessed neither strong antimicrobial activity nor did it induce acquired resistance. It protected crucifers from clubroot disease at 250  μ g mL⁻¹ following addition to the soil. Several antiauxins were tested for similar properties resulting in the suppression of clubroot disease and one, 2,3,5-triiodobenzoic acid, was effective at 10  μ g mL⁻¹. Clubroot reduction by epoxydon may result from antiauxin activity. This opens opportunities for a new group of agrochemicals.     

Effects of calcium on biocontrol activity of yeast antagonists against the postharvest fungal pathogen Rhizopus stolonifer

Calcium chloride (2% w/v) significantly inhibited the growth of the pathogen Rhizopus stolonifer , but did not affect the colony-forming units (CFU) of yeasts Candida guilliermondii and Pichia membranefaciens in potato dextrose broth. The concentration of yeast suspension influenced spore germination and germ tube growth of R. stolonifer in vitro, as well as disease incidence and lesion development in fruits. There were significant negative relationships between the suspension concentrations of the yeasts and the growth as well as infectivity of the pathogen. The addition of calcium resulted in lower spore germination rates and slower growth of germ tubes in vitro , as well as in lower disease incidences and smaller lesion diameters compared with treatments with yeast antagonists alone. When yeast cell suspensions reached a concentration of 5 × 10⁸ CFU mL⁻¹, growth of the pathogen was completely limited in vitro , and no infection was found in peach and nectarine fruits treated with or without calcium.     

Reduced sensitivity of Cercospora beticola isolates to sterol-demethylation-inhibiting fungicides

In a survey conducted during October 1995, single-lesion isolates of the sugar beet leaf-spot fungus, Cercospora beticola , were tested for sensitivity to the sterol demethylation inhibiting fungicides (DMIs) flutriafol and bitertanol. The isolates were collected from fields in three different areas of northern Greece. Fields at Serres and Imathia had been sprayed with DMIs for about 15 years to control sugar beet leaf-spot. At the third site, Amyndeon, DMI fungicides had not been used. From each area 150 isolates were tested. ED₅₀ values were calculated for individual isolates by regressing the relative inhibition of colony growth against the natural logarithm of the fungicide concentration. The mean ED₅₀ values for flutriafol for the Serres, Imathia and Amyndeon populations were 1·07, 0·73 and 0·5 µg mL⁻¹, respectively (significantly different at P  = 0·05). For bitertanol the mean ED₅₀ values for the Serres and Imathia populations were 0·72 and 0·81 µg mL⁻¹, respectively, which were not significantly different at P  = 0·05. The mean ED₅₀ value of the Amyndeon population was 0·48 µg mL⁻¹, which was significantly lower than those of the other two populations ( P  < 0·05). A cross-resistance relationship was found to exist between the two triazole fungicides tested when log transformed ED₅₀ values of 60 isolates were subjected to a linear regression analysis ( r  = 0·81).     

Berry infection and the development of bunch rot in grapes caused by Aspergillus carbonarius

The effects of different levels of inoculum of Aspergillus carbonarius and time of inoculation on berry infection and the development of aspergillus bunch rot on grapevines (cv. Sultana) were studied under field conditions. Inflorescences at full bloom were inoculated with aqueous spore suspensions of A. carbonarius containing 0 or 1 × 10⁶ spores mL⁻¹ in 2004/05 and 0, 1 × 10² or 1 × 10⁵ spores mL⁻¹ in 2005/06. In both years, the incidence of infection in inoculated berries was significantly higher than in uninoculated berries. Incidence of infection in berries from veraison until harvest was higher than at earlier stages of bunch development (berry set to berries that were still hard and green). Inoculation of bunches at veraison did not significantly increase A. carbonarius infection prior to harvest, at harvest, 6 days after harvest or when berries were over-ripe. Bunches inoculated at harvest did not significantly increase infection 6 days after harvest or when berries were over-ripe. Aspergillus carbonarius was isolated more frequently from the pedicel end (53·1%) than from the middle section (37·5%) and distal end (35·0%) of berries that were inoculated with 10⁵ spores mL⁻¹.     

Nematicidal activity of essential oils and organic amendments from Asteraceae against root-knot nematodes

The essential oil of Chrysanthemum coronarium flowerheads showed strong nematicidal activity in vitro and in growth-chamber experiments. Essential oil concentrations of 2, 4, 8 and 16  µ L mL⁻¹, significantly reduced hatch, J ₂ survival (determined by final value and area under curves of cumulative percentage hatch or mortality) and reproduction rate of Meloidogyne artiellia in vitro , with the lowest values occurring at 16  µ L mL⁻¹. In pot trials with chickpea cv. PV 61, essential oil concentrations of 10–40  µ L per 500 cm³ soil, applied on sterile cotton pellets, also significantly reduced the nematode's reproduction rate. The biological processes of mortality and hatching/reproduction were adequately described by the monomolecular and expanded negative exponential models, respectively. Effectiveness of soil amendment with either flowers, leaves, roots or seeds of C. coronarium , and flowers from several species of Asteraceae ( Chrysanthemum segetum , Calendula maritima , Calendula officinalis and Calendula suffruticosa ) at 5 g per 500 cm³ soil was tested for suppression of M. artiellia and growth of chickpea cv. PV 61 under growth-chamber conditions. In these tests, flowers of all five Asteraceae species and various parts of C. coronarium significantly reduced reproduction rates of M. artiellia , by 83·0–95·9%, with the minimum rates occurring in infected chickpea plants amended with flowers of C. officinalis and C. suffruticosa . The in vitro and in planta results suggest that the essential oil of C. coronarium and organic amendments from Asteraceae species may serve as nematicides.     

Control of Penicillium expansum by plant volatile compounds

Nine plant volatiles were tested for their activity in vitro and in vivo against Penicillium expansum , the cause of blue mould of pear. In vitro spore germination and mycelial growth assay showed a consistent fungicidal activity by trans -2-hexenal, carvacrol, trans -cinnamaldehyde and citral, while hexanal (-)- carvone, p -anisaldehyde, eugenol and 2-nonanone exhibited a progressively lower inhibition. trans -2-Hexenal was the best inhibitor of conidial germination [MIC (minimum inhibitory concentration) = 24·6  µ L L⁻¹; ED₅₀ = 10·2  µ L L⁻¹], while carvacrol was the best inhibitor of mycelial growth (MIC = 24·6  µ L L⁻¹; ED₅₀ = 9  µ L L⁻¹). The four most active compounds in in vitro studies were tested in vivo as fumigants against blue mould on pear cv. Conference. Best control was achieved by trans -2-hexenal vapour treatments (12·5  µ L L⁻¹) when applied over a 24-h period, beginning 24 h after inoculation. In contrast, carvacrol (12·5–200  µ L L⁻¹), and trans -cinnamaldehyde (50–400  µ L L⁻¹) were ineffective and citral (200  µ L L⁻¹) showed only slight effect.     

PCR-based specific and sensitive detection of Pectobacterium carotovorum ssp. carotovorum by primers generated from a URP-PCR fingerprinting-derived polymorphic band

A 24-mer primer pair was generated by sequencing a URP-PCR fingerprinting-derived polymorphic band that is uniquely shared in Pectobacterium carotovorum ssp . carotovorum strains (Pcc). The primer set (EXPCCF/EXPCCR) amplified a single band of expected size (0·55 kb) from genomic DNA obtained from 29 Pcc strains and three Pectobacterium carotovorum ssp. wasabiae (Pcw) strains, but not from other P. carotovorum subspecies atrosepticum , betavasculorum or odoriferum , or from other Erwinia spp. or bacterial genera. The Rsa I digestion profile of the amplified bands divided Pcc strains into five groups with a unique profile from Pcw strains. First-round PCR detected between 5 × 10² and 1 × 10³ colony forming units (CFU) mL⁻¹ and detection sensitivity was increased to as few as 2–4 CFU mL⁻¹ after second-round (nested) PCR. This PCR protocol was used directly to detect Pcc strains in infected plant tissues.     

Efficient, long-lasting resistance against the soft rot bacterium Pectobacterium carotovorum in calla lily provided by the plant activator methyl jasmonate

The potential of three externally applied chemical plant activators, Bion, BABA and methyl jasmonate, known to act only through the plant defence system and not on the pathogen directly, to induce resistance against wild-type Pectobacterium carotovorum was examined in white-flowered calla lily ( Zantedeschia aethiopica ). Following a 24-h induction period, plants were challenge-inoculated with P. carotovorum , originally isolated from calla lily or potato plants, previously transformed using a gfp broad-host-range promoter-probe vector. After another 24 h, Bion treatment (10  µ g mL⁻¹, as a drench) reduced disease symptoms more than sixfold and bacterial proliferation by four orders of magnitude. BABA treatment (5–10  µ g mL⁻¹, also as a drench) reduced the rate of infection by 75–85%. However, the protection afforded by both inducers did not persist. Also, at higher concentrations both displayed a phytotoxic effect. By contrast, methyl jasmonate (10 m m , applied as a leaf spray) completely inhibited P. carotovorum development in calla lily leaves and afforded a long-lasting effect. It is suggested that the defence response of calla lily against P. carotovorum involves the SA-signalling pathway in the short term, but the jasmonate/ethylene-signalling pathway is required for durable protection.     

Differential susceptibility of barnyard grass (Echinochloa crus-galli var. crus-galli) ecotypes to Exserohilum longirostratum

Ten barnyard grass ( Echinochloa crus-galli var. crus-galli ) ecotypes collected from several locations in Malaysian and Indonesian granaries were tested for variation in their susceptibility to the leaf blight pathogen ( Exserohilum longirostratum ). Four phenological growth stages of the ecotypes were sprayed with E. longirostratum at the rate of 1 × 10⁷ conidia mL⁻¹ under glasshouse conditions. The results of the study showed that plants inoculated with 1 × 10⁷ conidia mL⁻¹ and exposed for 24 h dew periods were severely infected and the percentage dry weight reduction of all ecotypes significantly increased. The 1–2 and 2–3-leaf stage plants were completely killed within 6 days after inoculation. However, susceptibility reactions among the ecotypes were observed at the 4–5-leaf stage, while the 6–7-leaf stage was resistant. The ecotypes, K-02, PK-04, KN-02, CJ-01, and L-01, were the most susceptible ecotypes as indicated by a higher area under disease progress curve value. The apparent disease progress rates for these ecotypes were significantly faster compared to the other ecotypes. The dry weight reduction among the four growth stages was variable. These findings show that the 1–2 and 2–3-leaf stages are the most susceptible, while the 6–7-leaf stage was resistant. This study has confirmed that a variable response to the fungal pathogen occurred within an intraspecific barnyard grass collection.     

Sensitivity of Phytophthora infestans to mandipropamid and the effect of enforced selection pressure in the field

Sensitivity to the new carboxylic acid amide (CAA) fungicide mandipropamid (MPD) in Phytophthora infestans was measured for isolates collected between 1989 and 2002 in Israel prior to the commercial use of MPD (baseline sensitivity, 44 isolates), and from MPD-treated (25 isolates) and untreated fields (215 isolates) in nine European countries and Israel between 2001 and 2005. All isolates were sensitive to MPD, with EC₅₀ values ranging between 0·02 and 2·98  µ g mL⁻¹. Plastic-tunnel (UK), shade-house (Israel) and field experiments (Israel) conducted during 2001–05 showed that enforced selection pressure, applied preventively or curatively, imposed by repeated sublethal (5  µ g mL⁻¹) or excessive (500–1000  µ g mL⁻¹) doses of MPD on mixed isolates of P. infestans produced no isolates resistant to the compound. The results of this study indicate that the probability of a buildup of resistant sub-populations of P. infestans to mandipropamid in the field is low.     

Factors influencing infection of eucalypts by Cylindrocladium pteridis

The pattern of Cylindrocladium pteridis adhesion, germination and penetration in eucalypt leaves was assessed using scanning electron microscopy. The effects of inoculum concentration, leaf wetness period, plant age and branch position of cylindrocladium leaf blight and defoliation severity were assessed in greenhouse studies using two Eucalyptus grandis × E. urophylla hybrid clones. Penetration occurred through stomata, and there was no difference in the number of penetrations between young and old leaves. Percentage leaf area with lesions and defoliation increased with the increase in inoculum concentration (1 × 10² to 10⁵ conidia mL⁻¹), duration of leaf wetness period (6 to 48 h) and plant age (60 to 180 days). Branch position in plants also significantly affected the percentage leaf area with lesions and defoliation, the latter variable being significantly higher at the stem base. The highest values of lesion area were also observed on leaves at the stem base in both clones. The Pearson correlation between defoliation and leaf area with lesions was significant in all experiments ( r  > 0·9) indicating a high association between these two variables.