Multiple-fungicide resistance to carbendazim,diethofencarb, procymidone,and pyrimethanil in field isolates of Botrytis cinerea from tomato in Henan Province,China

Botrytis cinerea, the fungal pathogen responsible for causing gray mold in tomatoes, frequently exhibits multiple fungicide resistance. In this study, the sensitivity to carbendazim (Car), diethofencarb (Die), procymidone (Prc), and pyrimethanil (Pyr) was determined for 263 isolates of B. cinerea from diseased fruit and leaves of tomato plants from 87 commercial greenhouses in Henan Province of China during 2013 and 2014. Of the isolates tested, 89%, 93%, 85%, and 86% were resistant to Car, Die, Prc, and Pyr, respectively. Twelve phenotypes were found having different sensitivity patterns, and multi-fungicide resistance was found to one, two, three or four fungicides. The percentage of multiple fungicide resistant isolates (Car^RDie^RPrc^RPyr^R) was 68%. Mycelial growth, spore production, and mycelial dry weight were not significantly different between resistant and sensitive phenotypes of the B. cinerea isolates. However, the virulence of Car^SDie^RPrc^SPyr^S isolates was higher than that of the other fungicide-resistance phenotypes. All of the carbendazim and diethofencarb resistant phenotypes carried a point mutation at codon 198 (E198A or E198K) or 200 (F200Y) in the β-tubulin gene, and all of the procymidone resistant phenotypes carried point mutations at codons 369 (Q369P) and 373 (N373S) in the Bos1 gene. These results provided important reference data for assessment of resistance risk of B. cinerea isolates from commercial tomato transplants in Henan Province, indicating the existence of a high frequency of resistance to carbendazim, diethofencarb, procymidone, and pyrimethanil revealing the necessity of a more integrated control of gray mold.     

Prevalence of isolates of Botrytis cinerea resistant to multiple fungicides in Chilean vineyards

Gray mold (Botrytis cinerea) is an important disease of grapevines (Vitis vinifera) and requires several fungicide treatments to achieve a satisfactory control in Chile. Furthermore, a high resistance risk has developed because of the extensive use of specific fungicides with a single-site mode of action. The aim of this study was to determine the presence of resistance to the multiple fungicides currently used against B. cinerea in Chile. During 2007–2009 and 2011, 214 isolates of B. cinerea from 36 commercial vineyards were examined for sensitivity to anilinopyrimidines (cyprodinil or pyrimethanil), demethylation inhibitors (DMIs) (tebuconazole), hydroxyanilides (fenhexamid) and phenylpyrroles (fludioxonil). Of all of the isolates, 62.7% were resistant to anilinopyrimidines, 7.2% to DMIs, 27.1% to hydoxyanilides and 44.8% to phenylpyrrole derivates. Overall, 16 (12.3%) of the isolates were sensitive; although none was resistant to all four of the fungicide classes, and 50 (38.5%), 51 (39.2%), and 13 (10.0%) isolates were resistant to one, two and three fungicides, respectively. In vitro, resistant isolates were capable of causing fruit rot on wounded apples pretreated with any one of the five fungicides at label concentrations. To the best of our knowledge, this is the first report of isolates having simultaneous resistance to anilinopyrimidines, DMIs, phenylpyrroles and hydroxyanilides in B. cinerea populations from grapevines in Chile. Therefore, fungicide resistance is a serious problem that questions the sustainability of the current gray mold control strategy, which relies almost exclusively on fungicides with single-site modes of action.     

Interface of the environment and occurrence of Botrytis cinerea in pre-symptomatic tomato crops

Botrytis cinerea (Grey mould) is a necrotrophic fungus infecting over 230 plant species worldwide. It can cause major pre- and post-harvest diseases of many agronomic and horticultural crops. Botrytis cinerea causes annual economic losses of 10–100 billion US dollars worldwide and instability in the food supply (Jin and Wu, 2015). Grey mould losses, either at the farm gate or later in the food chain, could be reduced with improved knowledge of inoculum availability during production. In this paper, we report on the ability to monitor Botrytis spore concentration in glasshouse tomato production ahead of symptom development on plants. Using a light weight and portable air sampler (microtitre immunospore trap) it was possible to quantify inoculum availability within hours. Also, this study investigated the spatial aspect of the pathogen with an increase of B. cinerea concentration in bio-aerosols collected in the lower part of the glasshouse (0.5 m) and adjacent to the trained stems of the tomato plants. No obvious relationship was observed between B. cinerea concentration and the internal glasshouse environmental parameters of temperature and relative humidity. However the occurrence of higher outside wind speeds did increase the prevalence of B. cinerea conidia in the cropping environment of a vented glasshouse. Knowledge of inoculum availability at time periods when the environmental risk of pathogen infection is high should improve the targeted use and effectiveness of control inputs.     

Improvement of Hanseniaspora uvarum biocontrol activity against gray mold by the addition of ammonium molybdate and the possible mechanisms involved

The efficacy of Hanseniaspora uvarum against gray mold by adding ammonium molybdate (NH₄–Mo) and the mode of actions were evaluated. The results showed that H. uvarum at 1 × 10⁶ CFU ml⁻¹ plus 1 mmol l⁻¹ NH₄–Mo greatly reduced gray mold in grape fruits. NH₄–Mo at concentrations of 1, 5, 10 and 15 mmol l⁻¹ significantly inhibited spore germination and mycelium growth of Botrytis cinerea. Population growth of H. uvarum was markedly inhibited by NH₄–Mo at 5 mmol l⁻¹in vitro and not affected by addition of NH₄–Mo at 1 and 5 mmol l⁻¹ in wounds combination of NH₄–Mo and H. uvarum induced higher activities of peroxidase (POD), polyphenoloxidase (PPO), phenylalanine ammonialyase (PAL), superoxide dismutase (SOD), catalase (CAT) and β-1,3-Glucanase than individual application of H. uvarum or NH₄–Mo. The enhancement of disease control may be directly because of the inhibitory effects of NH₄–Mo on spore germination and mycelial growth of B. cinerea in vitro, and indirectly because of the induced defense reactions by NH₄–Mo in grape berries.     

Baseline sensitivity of Monilinia laxa from Greece to fenhexamid and analysis of fenhexamid-resistant mutants

Fenhexamid is a hydroxyanilide fungicide with excellent performance against Botrytis cinerea but also effective against Monilinia spp. which cause brown rot disease in apple and stone fruit. A total of 75 Monilinia laxa field isolates were utilized to determine baseline sensitivity while a number of fenhexamid-resistant laboratory mutants were used to evaluate the resistance risk associated with the longevity of the effectiveness of fenhexamid. Fenhexamid was found to be highly effective against all field isolates. EC₅₀ values ranged from 0.02 to 1 μg mL⁻¹ and were distributed unimodaly around an average of 0.1 μg mL⁻¹. M. laxa laboratory strains with moderate and high resistance levels to fenhexamid were isolated after UV mutagenesis. All fenhexamid-resistant strains showed parental sensitivity to carbendazim, iprodione, fludioxonil, pyraclostrobin, flusilazole and prochloraz. Interestingly, some of the mutant strains were also resistant to tridemorph, fenpropimorph and spiroxamine. Studies on fitness parameters of fenhexamid-resistant strains revealed a fitness cost on sporulation and pathogenicity but not on mycelial growth. These results suggest that fenhexamid should be a good alternative site-specific fungicide for the control of brown rot disease caused by M. laxa. However, appropriate anti-resistance strategies should be considered to ensure the successful commercial use of fenhexamid in the long run.     

Effects of 1,3-dichloropropene on nematode,weed seed viability and soil-borne pathogen

1,3-dichloropropene (1,3-D, C₃H₄Cl₂) is one of the potential candidates as soil disinfectant since the restriction of methyl bromide (MeBr) in soil fumigation due to its ecological risk. Its nematode, soil-borne pathogen and weed control efficacies were evaluated in a laboratory dose-response study and in two commercial tomato fields. Laboratory studies found that the seeds of Digitaria chinensis Hornem. were the most sensitive to soil fumigation with 1,3-D, followed by Eleusina indica (Linn.) Gaertn., Echinochloa crusgalli (L.) Beauv. and Amaranthus retroflexus L. with the LC₉₀ values between 14.23 and 73.59 mg kg⁻¹ soil. Among the pathogens, Phytophthora capsici Leonian was the most sensitive and Fusarium oxysporum f. sp. fragariae was the least sensitive to 1,3-D fumigation with the LC₅₀ values were 0.24 and 1.55 g m⁻². Rhizoctonia solani Kühn., Phytophthora nicotianae var. nicotianae and Botrytis cinerea Persoon exhibited intermediate susceptibility. Field trials revealed that 1,3-D applied to the field at 180, 120 and 80 L ha⁻¹ could suppress Meloidogyne incognita root galling while maintaining high tomato marketable yields, better than Dazomet at the concentration of 400 kg ha⁻¹. Our results indicated that 1,3-D was an excellent nematicide and could provide good to moderate weed and pathogen control. Based on our results, 1,3-D, in combination with other alternatives to MeBr is recommended to reach an integrated pest management.     

Physiology of yield determination of mung bean (Vigna radiata (L.) Wilczek) under various irrigation regimes in the dry and intermediate zones of Sri Lanka

Drought is a major factor limiting yield improvement of mung bean (Vigna radiata (L.) Wilczek) in the sub-humid, dry and intermediate zones of Sri Lanka. Therefore, the objective of this study was to analyze the yield response of mung bean to irrigation at various phenological stages in terms of radiation interception, radiation-use efficiency and harvest index. Four field experiments were carried out at two sites (Maha-Illuppallama and Kundasale) during the short, dry yala season over two years (1995 and 1996). The life cycle of mung bean was divided into three stages: vegetative (from germination to appearance of first flower); flowering (from appearance of first flower to 75% pod initiation); and pod-filling (from 75% pod initiation to maturity). Eight irrigation treatments were defined as all possible combinations of irrigation during the three stages. Maximum potential soil water deficits (PSWD) ranging from 127 to 376 mm developed as a result of keeping different combinations of stages unirrigated. Maximum LAI (L_m) and the fraction of incoming radiation intercepted (F) increased significantly with the number of stages irrigated. Specifically, treatments which included irrigation during the vegetative stage achieved large L_m and F. Radiation-use efficiency (RUE), maximum total biomass (W_m), harvest index (HI) and seed yield (Y) also showed a significant positive response to the number of stages irrigated. However, all the above parameters were significantly greater in treatments which included irrigation during the pod-filling and flowering stages. The treatment which received irrigation only during the vegetative stage had significantly lower RUE, W_m, HI and Y despite having higher L_m and F. Therefore, irrigation is critical during pod-filling and flowering stages mainly because of the higher LAI during these periods and, consequently, the greater demand for water. Lack of irrigation during these critical stages resulted in the development of significant PSWD with adverse effects on photosynthesis and consequently decreased RUE. Moreover, water stress during flowering and pod-filling stages significantly reduced pod initiation and pod growth rates and thereby reduced HI. It is concluded that to maximize mung bean yields in the dry season of the sub-humid zones of Sri Lanka, irrigation should extend across all phenological stages, specially the pod-filling stage.     

Evaluation of electrolyzed oxidizing water for phytotoxic effects and pre-harvest management of gray mold disease on strawberry plants

Near neutral (pH = 6.3-6.5) electrolyzed oxidizing water (EO water) has been demonstrated to inactivate fungi in pure culture and to mitigate infection on fruit surfaces. One possible alternative or supplement to traditional pre-harvest crop management practices that currently rely on the use of large quantities of fungicides is near neutral EO water. In the present work, treatment of Botrytis cinerea or Monilinia fructicola with near neutral EO water (50 or 100 ppm total residual chlorine (TRC)) in pure culture resulted in a 10⁶ reduction and 100% inactivation as evidenced by negative broth enrichment. When applied in concert with 50 or 100 ppm EO water, treatments of Captan 50WP (captan), Rovral (iprodione), Iprodione 4LAG (iprodione), or Switch 62.5 WDG (cyprodinil and fludioxonil) effectively inhibited fungal growth of B. cinerea as evidenced by a 10⁶ reduction on the direct plate and negative broth enrichment. Treatments of Captan 50WG (captan), Rovral (iprodione), Iprodione 4LAG (iprodione), Switch 62.5 WDG (cyprodinil and fludioxonil), Captan 80 WDG (captan), or Captevate (captan and fenhexamide) when applied in concert with 50 or 100 ppm EO resulted in a 10⁶ reduction of M. Fructicola and 100% inactivation as evidenced by negative broth enrichment. Strawberry plants sprayed with EO water (pH = 6.3-6.5) at concentrations of 50 and 100 ppm TRC once per week, did not result in significant (P > 0.05) phytotoxicity relative to a water (0 ppm TRC) treatment. In this study, the application of 100 ppm EO water (pH = 6.3-6.5) twice per week to strawberry plants infected with B. cinerea was more effective (P ≤ 0.05) than a once per week Captan application and as effective as a once per week captan/once per week EO treatment. The once per week captan/once per week EO treatment was significantly more effective (P ≤ 0.05) than the captan once per week treatment. Dip treatments of strawberries in near neutral EO solutions (50 and 100 ppm TRC; pH = 6.3-6.5) did not leave a chlorine residue on the fruit relative to a water dip. The results from this study suggest that near neutral EO solutions could be used to manage infection of B. cinerea on strawberry plants in the field and also as a disinfection solution for harvesting equipment, greenhouses, packing houses and in commercial facilities to prevent or manage infections of B. cinerea and M. fructicola.     

Antifungal activities of extracts produced by liquid fermentations of Chilean Stereum species against Botrytis cinerea (grey mould agent)

Extracts obtained from liquid mycelial fermentations of Chilean species of the Stereum genus showed antifungal activity against Botrytis cinerea. Thirty-six strains were evaluated in vivo and in vitro assays, 25 belonging to Stereum hirsutum (Sh) and 11 to Stereum rameale (Sr). Two types of extracts were obtained: EtOAc-extract (liquid phase) and MeOH-extract (mycelial phase). Plate diffusion assay showed that EtOAc-extracts were more active than MeOH-extracts. S. hirsutum included 4 strains with the highest antifungal activity (Sh134-11, Sh144-11, Sh152-11, Sh155-11) and S. rameale highlighted with two strains (Sr25-11, Sr27-11). Effects on the mycelial growth of B. cinerea showed that EtOAc-extracts produced by S. hirsutum (Sh134-11, Sh152-11) inhibited from 1000 μg mL⁻¹, reaching 67 and 49%, respectively. At 2000 μg mL⁻¹, these strains inhibited nearly 80% of mycelial growth. EtOAc-extract of Sh134-11 was more effective to control the sporogenesis, inhibiting in 100% the sporulation at 500 μg mL⁻¹. Assays showed that Sh134-11 and Sh152-11 exhibited a minimal fungicidal concentration (MFC) of 50 and 100 μg mL⁻¹ respectively and minimal inhibitory concentration (MIC) at 20 μg mL⁻¹. EtOAc-extracts of Sr25-11 and Sr27-11 showed MFC value at 100 μg mL⁻¹ and MIC at 20 and 50 μg mL⁻¹, respectively. Strawberries treated with 1000 μg mL⁻¹of Sh134-11 and Sr25-11 reached 82 and 72% of decay inhibition, respectively. Treatments with 2000 μg mL⁻¹ showed a decay inhibition of 90% approximately. In vivo tests are in accordance with the results obtained in vitro assays, confirming the efficacy of Sh134-11 and Sr25-11 to control B. cinerea. Differences in antifungal activities observed in the different strains suggested that the ability to produce bioactive compounds is not homogenously distributed among S. hirsutum and S. rameale. Our study would suggest that submerged fermentations of Chilean S. hirsutum strain Sh134-11 produce extracts, which could be used as possible biofungicides and an alternative to synthetic fungicides.     

Bis(2,3-dibromo-4,5-dihydroxybenzyl) Ether,a Marine Algae Derived Bromophenol,Inhibits the Growth of Botrytis cinerea and Interacts with DNA Molecules

Bis(2,3-dibromo-4,5-dihydroxybenzyl) ether (BDDE) is a bromophenol isolated from marine algae. Previous reports have shown that BDDE possesses cytotoxic and antibacterial activity. In the present study, we demonstrate that BDDE displays broad-spectrum antifungal activities, especially on Botrytis cinerea. BDDE inhibits the growth of B. cinerea cultured on a solid medium of potato dextrose agar (PDA) as well as on the potato dextrose broth (PDB) medium. Moreover, BDDE decreases the incidence of fruit decay and severity of strawberries infected with B. cinerea. Further studies have revealed that BDDE decreases the germination rate and inhibits the mycelial growth of B. cinerea. The inhibition mechanisms are related to the disruption of the cell membrane integrity in B. cinerea spores and newly formed germ tubes. This study also suggests that BDDE possibly interacts with DNA via intercalation and minor groove binding. The studies provide evidence that BDDE has potential application in the control of gray mold after fruit harvest and the compound could serve as a candidate or lead template for rational drug design and for the development of antifungal agents.     

Antifungal activity of polyacetylenes isolated from Cirsium japonicum roots against various phytopathogenic fungi

Antifungal substances from a methanol extract of Cirsium japonicum roots were purified and characterized, and their antifungal activities against various plant pathogens were evaluated. Three polyacetylene substances were isolated from roots of C. japonicum using repeated column chromatography; these were identified as ciryneol A, ciryneol C and 1-heptadecene-11,13-diyne-8,9,10-triol by mass and nuclear magnetic resonance spectral analyses. In vitro antifungal activity of the three substances varied according to compound and target species. Magnaporthe oryzae, Colletotrichum coccodes, Colletotrichum acutatum, Pythium ultimum and Botrytis cinerea were relatively sensitive to the three polyacetylenes, with IC₅₀ values below 50 μg mL⁻¹. In vivo, they all showed similar and broad antifungal spectra against the seven plant diseases tested. At 500 μg mL⁻¹, all three compounds effectively suppressed the development of rice blast, rice sheath blight, tomato late blight, wheat leaf rust and red pepper anthracnose, with control values over 90%. They were highly active especially against wheat leaf rust; they controlled the development of this disease more than 88% even at a concentration of 125 μg mL⁻¹. In addition, ciryneol C effectively suppressed barley powdery mildew. This is the first report on the antifungal activities of the three polyacetylenes from roots of C. japonicum against plant pathogenic fungi. Polyacetylenes from roots of C. japonicum may contribute to the development of environmentally safer alternatives to protect crops from various phytopathogenic fungi.     

Fungicidal activity of fluopicolide for suppression of Phytophthora capsici on squash

The efficacy of a new fungicide fluopicolide in suppression of Phytophthora blight caused by Phytophthora capsici was evaluated under laboratory and field conditions. Studies with 51 P. capsici isolates from vegetable crops in Georgia, USA, indicated that 5.9% of the isolates were resistant, 19.6% were intermediately sensitive, and 74.5% were sensitive to 100 μg ml⁻¹ of mefenoxam based on in vitro mycelial growth. EC₅₀ values of fluopicolide in inhibiting mycelial growth of 25 isolates, representing resistant, intermediately sensitive, and sensitive to mefenoxam, ranged from 0.05 to 0.35 μg ml⁻¹ with an average of 0.2 μg ml⁻¹ EC₅₀ values of fluopicolide in suppressing zoospore germination and sporangium production of the 25 isolates ranged from 1.1 to 4.5 μg ml⁻¹ and 0.3–9.0 μg ml⁻¹, respectively. Evaluation of a collection of 150 P. capsici isolates from vegetables and irrigation ponds found none of the isolates were resistant to 10 μg ml⁻¹ of fluopicolide. Field experiments were conducted to determine the efficacy and application methods of fluopicolide for control of P. capsici on squash in spring 2007 and 2009. Fluopicolide applied through drip irrigation or as a foliar spray at 86.6 or 115.4 g ha⁻¹ consistently provided significant disease reduction and increased squash yield. Results with fluopicolide were similar or slightly superior to mefenoxam applied at recommended rate. Fluopicolide applied at 57.7 g ha⁻¹ did not provide consistent satisfactory disease suppression. The results indicated that fluopicolide was effective in suppression of different stages of the life cycle of P. capsici and could be a viable alternative to mefenoxam for managing Phytophthora blight in squash production.     

Hazard from reliance on cruciferous hosts as sources of major gene-based resistance for managing blackleg (Leptosphaeria maculans) disease

The host range of eighty isolates of Leptosphaeria maculans obtained from various cruciferous hosts in Western Australia was tested by inoculating onto cotyledons of 13 cruciferous hosts, including six Brassica species and one interspecific line of Brassica. These field isolates produced highly variable responses across the hosts tested. One or more isolates overcame the resistance in each of the tested Brassica species with the B-genome, such as B. juncea, B. nigra and B. carinata and of other species tested, including Raphanus raphanistrum, Crambe abyssinica, Sinapis alba, Eruca vesicaria and Raphanus sativus. None of the isolates tested to date were virulent on Camelina sativa. Of all isolates, 48.8% (and 75% of isolates taken from cultivars containing single dominant gene-based resistance derived from B. rapa ssp. sylvestris) showed high virulence against canola cultivars containing this single dominant gene-based resistance. There was a relationship between host source of isolates with virulence. Results clearly illustrated that extensive levels of variation exist within L. maculans populations in Western Australia such that major gene-based resistance in a range of these hosts could be rapidly overcome by one or more field populations of the pathogen. It is noteworthy that this ability to overcome such resistance in various cruciferous hosts was found to be present in certain isolates in the L. maculans population even prior to exposure of the pathogen to such major gene-based resistance sources in commercial crops. Our results indicated that seeking major gene-based resistance from within cruciferae for B. napus canola breeding, including from taxa which to date have shown resistance to L. maculans when used in the field as rotation, industrial oil or green manure crops, may lead to both breakdown and wastage of these valuable sources of resistance to blackleg disease.     

Using legumes to enhance nitrogen fertility and improve soil condition in cotton cropping systems

Many Australian cotton growers now include legumes in their cropping system. Three experiments were conducted between 1994 and 1997 to evaluate the rotational effects of winter or summer legume crops grown either for grain or green manuring on following cotton (Gossypium hirsutum L.). Non-legume rotation crops, wheat (Triticum aestivum) and cotton, were included for comparison. Net nitrogen (N) balances, which included estimates of N associated with the nodulated roots, were calculated for the legume phase of each cropping sequence. Faba bean (Vicia faba — winter) fixed 135–244 kg N ha⁻¹ and soybean (Glycine max — summer) fixed 453–488 kg N ha⁻¹ and contributed up to 155 and 280 kg fixed N ha⁻¹, respectively, to the soil after seed harvest. Green-manured field pea (Pisum sativum — winter) and lablab (Lablab purpureus — summer) fixed 123–209 and 181–240 kg N ha⁻¹, respectively, before the crops were slashed and incorporated into the topsoil.In a separate experiment, the loss of N from ¹⁵N-labelled legume residues during the fallow between legume cropping and cotton sowing (5–6 months following summer crops and 9 months after winter crops) was between 9 and 40% of ¹⁵N added; in comparison, the loss of ¹⁵N fertilizer (urea) applied to the non-legume plots averaged 85% of ¹⁵N added. Little legume-derived ¹⁵N was lost from the system during the growth of the subsequent cotton crop.The improved N fertility of the legume-based systems was demonstrated by enhanced N uptake and lint yield of cotton. The economic optimum N fertilizer application rate was determined from the fitted N response curve observed following the application of N fertilizer at rates between 0 and 200 kg N ha⁻¹ (as anhydrous ammonia). Averaged over the three experiments, cotton following non-legume rotation crops required the application of 179 kg N ha⁻¹, whilst following the grain- and green-manured legume systems required only 90 and 52 kg N ha⁻¹, respectively.In addition to improvements in N availability, soil strength was generally lower following most legume crops than non-legume rotation crops. Penetrometer resistance during the growth of the subsequent cotton crop increased in the order faba bean, lablab, field pea, wheat, cotton, and soybean. It is speculated that reduced soil strength contributed to improvement in lint yields of the following cotton crops by facilitating the development of better root systems.     

Use of essential oil of Laurus nobilis obtained by means of a supercritical carbon dioxide technique against post harvest spoilage fungi

The aspects of the antifungal activity of essential oil of laurel (Laurus nobilis) obtained by means of a supercritical carbon dioxide (SFE-CO₂) technique against post harvest spoilage fungi, have been studied in this research work by tests performed under in vitro and in vivo conditions. The measurement of antifungal activity of the oil, for its potential application as botanical fungicide, is very useful to find alternatives to synthetic fungicides. The present paper reports, for the first time, the results about the antifungal activity of laurel oil, obtained by a semi-industrial process that utilize a SFE-CO₂ technique, against three plant pathogenic fungi. The determination of the main active substances was carried out by gas chromatography analysis: laurel oil was characterized by high content (≥10%) of 1.8-cineole, linalool, terpineol acetate, methyl eugenol and a low content (<10%) of linalyl acetate, eugenol, sabinene, β-pinene, α-terpineol. The inhibition of the mycelial growth of Botrytis cinerea, Monilinia laxa and Penicillium digitatum was evaluated in vitro at the concentration range of 200, 400, 600, 800 and 1000 μg/mL. M. laxa was totally inhibited by application of the oil at the lowest concentration, B. cinerea was completely inhibited at the highest concentration, and a fungistatic action was observed in both cases. P. digitatum was only partially inhibited at all the concentration ranges. The activity of the oil, placed in the form of spray on the fruit skin at the concentration range of 1, 2 and 3 mg/mL, was studied by biological tests. Both curative and protective activities of the oil have been evaluated on peaches, kiwifruits, oranges and lemons artificially inoculated with M. laxa, B. cinerea and P. digitatum, respectively. A very good antifungal activity has been found on kiwifruits and peaches when the oil was placed before the inoculation at a concentration of 3 mg/mL (68 and 91% of decay inhibition respectively). The same activity has been found on peaches when the oil was placed after the infection (76% of decay inhibition). The application of the oil did not caused any phytotoxic effect and kept any fruit flavour, fragrance or taste. This study has demonstrated that the essential oil of L. nobilis extracted by a SFE-CO₂ technique, is one potential and promising antifungal agent which could be used as botanical fungicide in the postharvest protection of peaches and kiwifruits against M. laxa and B. cinerea.     

Antifungal activity of liquid waste obtained from the detoxification of steam-exploded plant biomass against plant pathogenic fungi

The antifungal activity of steam-exploded liquid waste (SELW) produced by the detoxification of steam-exploded biomass of Miscanthus sinensis, Arundo donax and wheat straw for 2nd generation industrial bioethanol production was studied against plant pathogenic fungi for the first time. Quantification of fermentation inhibitors (2-furaldehyde, 5-hydroxymethylfurfural, acetic and formic acid) was carried out by standard methods. Mycelial growth inhibition and conidial germination of eight fungal strains [Alternaria alternata, Botrytis cinerea, Colletotrichum acutatum, Cladosporium fulvum, Fusarium oxysporum f. sp. lycopersici (FOL), F. oxysporum f. sp. melonis (FOM), F. solani f. sp. pisi and Verticillium dahliae] were evaluated in vitro at different dilution rates (1:2, 1:4 and 1:8) for each SELW. Disease suppressiveness was assessed in vivo in eight horticultural pathosystems (A. alternata/tomato, B. cinerea/tomato, C. acutatum/strawberry, C. fulvum/tomato, FOL/tomato, FOM/melon, F. solani f. sp. pisi/pea and V. dahliae/eggplant) under greenhouse conditions. Pathogen suppression by each SELW, applied both in spray form on to tomato leaves and skins of tomato and strawberry, and by means of dipping method in watering suspensions on to seedling root systems of tomato, melon, pea and eggplant, was evaluated at dilution rates of 1:2, 1:4 and 1:8 using irrigation water during curative and preventive treatments. Investigations carried out in vitro showed that M. sinensis SELW was more suppressive than wheat straw SELW, and A. donax SELW was less suppressive than wheat straw SELW at the lowest dilution rates. Relationships between the concentration of 2-furaldehyde, acetic and formic acid present in SELWs and their antifungal effect were found. Moreover, B. cinerea, C. fulvum, V. dahliae, A. alternata, C. acutatum and F. solani f. sp. pisi were more effectively inhibited than FOL and FOM. Investigations performed in vivo showed that SELWs of M. sinensis and wheat straw can be diluted at a 1:2 rate and used for controlling six fungal diseases during preventive treatments. In particular, they can be used by dipping of the root systems during transplanting in the case of F. solani f. sp. pisi/pea and V. dahliae/eggplant; on the other hand, they can be nebulized on to the leaves and fruits before symptoms appearance in the case of A. alternata/tomato, B. cinerea/tomato, C. acutatum/strawberry and C. fulvum/tomato. This study is of particular interest because it points out how these SELWs could be employed in horticultural crop protection in Southern Italy, thereby making it possible to effectively combine industrial production of 2nd generation biofuels with sustainable horticulture under greenhouse conditions.     

Characterization of fungicide resistant isolates of Phaeoacremonium aleophilum infecting grapevines in Spain

Fungicide resistance in Phaeoacremonium aleophilum, one of the most frequent fungal pathogens associated with grapevine trunk diseases, was investigated and found to exist in some isolates of the pathogen against a commercial formulation, Escudo^®. The effect of this compound and its two active substances, carbendazim and flusilazole, was first evaluated on the mycelial growth of P. aleophilum. Escudo^®-resistant isolates were estimated at a frequency of 24% in Spanish vineyards. Then, the two active substances were used individually to test their effect on mycelia growth of twelve single-spore cultures originating from six Escudo^®-resistant isolates. Flusilazole (DMI-triazole) did not inhibit mycelia growth of any single-spore cultures of P. aleophilum. Carbendazim (benzimidazole) used alone allowed the growth of the same single-spore cultures that were also resistant to Escudo^®. AFLP characterization of sensitive and resistant single-spore cultures showed genetic diversity within P. aleophilum isolates but no AFLP markers were associated with resistance. New primers set (L2/R1) were designed to partially amplify the exon 6 of the beta-tubulin gene of P. aleophilum. Two different point mutations resulted in glycine (GGC) or lysine (AAA) replacing the glutamic acid (GAG) at codon 198 of the beta-tubulin gene in some of the resistant single-spore cultures studied. Resistant single-spore cultures of P. aleophilum were shown to have different aggressiveness levels as sensitive single-spore cultures by inoculation of wood segments of Vitis vinifera in the presence and absence of fungicide.     

Management of Meloidogyne incognita in yam-based cropping systems with cover crops

The effect of intercropping cover crops was with yam was assessed for nematode management both in pot and field conditions in Nigeria. The cover crops were sown with yams in pots containing sterile soil and inoculated with 5000 eggs of Meloidogyne incognita. In the field, each cover crop was separately intercropped with yams inoculated with 10,000 nematodes. From both pot and field experiments, no nematode damage was observed on yam tubers that were intercropped with Aeschynomene histrix, Crotolaria juncea, and Tagetes erecta. Damage in intercropped tubers was reduced by 72.7% with Mucuna pruriens, Centrosema pubescens, and Pueraria phaseoloides and by 58.3% with Stylosanthes guianensis compared to yams planted without cover crops; they also had lower (p ≤ 0.05) nematode populations. Cajanus cajan, Lablab purpureus and Vigna unguiculata however, supported high nematode populations and led to nematode damage in intercropped yams. Tubers from M. incognita-inoculated plants were more damaged and lost 42% more weight (p ≤ 0.05) following three months of storage than tubers from uninoculated plants. Intercropping yams with selected cover crops can be useful in managing M. incognita without reducing yam yields.     

Assessment of rhizobacteria from grapevine for their suppressive effect on the parasitic nematode Xiphinema index

The capacity of rhizobacteria isolated from grape plants to protect grapevine roots from damage by the nematode Xiphinema index was assessed in trials carried out in the growing season 2008–2009 and 2009–2010. A total of two groups of bacteria were evaluated, one comprising 49 isolates, of which 37 were assessed in previous studies, and one comprising 90 isolates not previously evaluated. Two assays were performed on the first group, one per season, and one on the second group, lasting two growing seasons. Pots of field soil (3 L) planted with two-month old Thompson Seedless vines were inoculated with 200 or 400 specimens of X. index after inoculation with 10⁶ cfu/ml of test rhizobacteria; the posts were maintained in a shaded greenhouse. Seven isolates from the first group of bacteria (Bacillus brevis, Bacillus megaterium, Cytophaga johnsonae, Pseudomonas fluorescens, Rahnella aquatilis and Stenotrophomonas maltophilia) were effective in decreasing damage in both of the assays (P < 0.05). Ten isolates from the second group (Variovorax paradoxus, Pseudomonas pseudoalcaligenes, Bacillus mycoides, Bacillus sphaericus, Bacillus thuringiensis, Curtobacterium flaccumfaciens, Pseudomonas putida, Pseudomonas alcaligenes and Pseudomonas viridiflava) resulted in the lowest number of galls per gram root. B. brevis and B. megaterium also demonstrated good activity in previous studies on grape plants and are thus promising candidates for further research.     

Effect of nitrogen supply on crop conductance,water- and radiation-use efficiency of wheat

Water-use efficiency (WUE_DM) is directly related to radiation-use efficiency (RUE) and inversely related to crop conductance (g_c). We propose that reduced WUE_DM caused by shortage of nitrogen results from a reduction in RUE proportionally greater than the fall in conductance. This hypothesis was tested in irrigated wheat crops grown with contrasting nitrogen supply; treatments were 0, 80 and 120 kg N ha⁻¹ in 1998 and 0, 80, 120 and 160 kg N ha⁻¹ in 1999. We measured shoot dry matter, yield, intercepted solar radiation and soil water balance components. From these measurements, we derived actual evapotranspiration (ET), soil evaporation and transpiration, WUE_DM (slope of the regression between dry matter and ET), WUE_Y (ratio between grain yield and ET), RUE (slope of the regression between dry matter and intercepted radiation), and g_c (slope of the regression between transpiration and intercepted radiation). Yield increased from 2.3 in unfertilised to an average 4.7 t ha⁻¹ in fertilised crops, seasonal ET from 311 to 387 mm, WUE_DM from 23 to 37 kg ha⁻¹ mm⁻¹, WUE_Y from 7.6 to 12.4 kg ha⁻¹ mm⁻¹, RUE from 0.85 to 1.07 g MJ⁻¹, while the fraction of ET accounted for soil evaporation decreased from 0.20 to 0.11. In agreement with our hypothesis, RUE accounted for 60% of the variation in WUE_DM, whereas crop conductance was largely unaffected by nitrogen supply. A greater fraction of evapotranspiration lost as soil evaporation also contributed to the lower WUE_DM of unfertilised crops.