Effect of Trichoderma asperellum and arbuscular mycorrhizal fungi on cacao growth and resistance against black pod disease

Arbuscular mycorrhizal fungi (AMF) and members of the genus Trichoderma have emerged as promising groups of microbial inoculants that can induce plant growth and resistance to disease. This study aimed at investigating the potential of AMF and a strain (PR11) of Trichoderma asperellum to promote cacao growth and induce resistance against Phytophthora megakarya. Cacao seedlings were either non-inoculated, or inoculated with the saprophytic fungus T. asperellum and/or a mixture of two different mycorrhizal fungi, Gigaspora margarita and Acaulospora tuberculata. Eighteen weeks after planting, a series of morphological as well as biochemical changes, which are considered to be part of the plant defense response, were measured after a challenge inoculation of the leaves with zoospores of P. megakarya. Inoculation with AMF and T. asperellum alone was essential for the promotion of plant growth. Significant increase in plant height, root and shoot fresh weights, as well as phosphorous uptake was recorded in comparison to non-inoculated control plants. However, dual inoculation of cacao seedlings with T. asperellum and AMF did not always positively benefit the plants. Leaf inoculation showed variation among the treatments, with the lowest disease index (highest level of resistance) recorded in plants inoculated with either AMF or T. asperellum only. This came along with a high synthesis of amino acids and phenolic compounds in both healthy and infected leaves, suggesting that these metabolites are implicated in disease resistance.     

Actinomycetes mediated biochemical responses in tomato (Solanum lycopersicum) enhances bioprotection against Rhizoctonia solani

Six actinomycetes isolates, namely Streptomyces toxytricini vh6, Streptomyces flavotricini vh8, S. toxytricini vh22, Streptomyces avidinii vh32, Streptomyces tricolor vh85 and vh41, an isolate of an unknown species of Actinomycetales, were tested for their efficacy in protecting tomato (Solanum lycopersicum) against Rhizoctonia solani under green house conditions. Actinomycetes treated plants showed better growth in terms of high chlorophyll content, higher phenylalanine ammonia lyase (PAL) activity and high total phenolic content. Qualitative and quantitative estimation of phenolic compounds from tomato leaves showed significant accumulation of six phenolic acids, gallic (29.02 μg g⁻¹ fresh leaf wt), ferulic (11.44 μg g⁻¹ fresh wt), cinnamic (56.84 μg g⁻¹ fresh wt), gentisic (24.19 μg g⁻¹ fresh wt), chlorogenic acid (1.72 μg g⁻¹ fresh wt) and salicylic (0.39 μg g⁻¹ fresh wt) acid, in actinomycetes treated plants. Biochemical profiling, when correlated with plant mortality in actinomycetes treated and untreated plants, indicated that isolates vh6 and vh8 offered 44.55% and 40.14% disease reductions, respectively compared to the control. These results established that these organisms have the potential to act as biocontrol agents.     

Effects of middle-viscosity chitosan on Ramularia cercosporelloides

The inhibitory properties of middle-viscosity Chitosan on the growth of fungus Ramularia cercosporelloides were studied in vitro. The inhibitory concentration that delayed 50% of the radial growth (IC₅₀) was 3.4 g l⁻¹ for middle-viscosity Chitosans with molecular weights of about 133 and 187 kDa (middle molecular weight) dissolved in lactic and acetic acids, respectively. At 96 h of incubation and under the same growing conditions, inhibitions of 91.79% and 73.13% of the radial growth of the fungus were observed when 3.4 g l⁻¹ of Chitosan was dissolved in 0.05 M acetic and 0.05 M lactic acid, respectively. The biomass production was significantly lower than that observed in the controls at 72 h. Based on these in vitro results; Chitosan could be a good alternative to control the disease caused by R. cercosporelloides on safflower.     

Biocontrol of Rhizoctonia solani and Sclerotium rolfsii on tomato by delivering antagonistic bacteria through a drip irrigation system

In a 2-year assessment carried out on tomato crops of Central and Southern Italy, a high incidence of Rhizoctonia solani and Sclerotium rolfsii was found. These fungal pathogens attack horticultural crops and are responsible for severe crown and stem rot. Because of technical, economical and environmental issues their chemical control is an arduous task. To find alternative and eco-compatible control methods, the effectiveness of two new antagonistic bacterial isolates (Burkholderia cepacia, T1A-2B, and Pseudomonas sp., T4B-2A), previously selected from suppressive organic amendments, were tested on tomato plants grown under both growth chamber and field conditions. The potential antagonists were compared with two commercial biofungicides, based on Bacillus subtilis (BSF4) and Trichoderma asperellum (TV1), and four synthetic fungicides (tolclofos-methyl, azoxystrobin, fosetyl-Al and fosetyl-Al + propamocarb). In 2-year field experiments carried out on tomato plants, the biocontrol bacteria as well as the other treatments were applied to the soil, proximal to the plant crowns and main roots, by means of an effective and specific system of drip irrigation. In all the experiments the novel selected biocontrol bacteria significantly reduced both incidence and severity of the diseases caused by S. rolfsii or R. solani, with results demonstrating effectiveness equal to TV1, better than BSF4 and comparable with the synthetic fungicides, except for tolclofos-methyl which was the most effective treatment. In field experiments, carried out for two consecutive years, isolate T1A-2B reduced up to 58.33% and up to 63.8% the severity of the diseases caused by S. rolfsii and R. solani respectively; whereas isolate T4B-2A gave reduction of S. rolfsii and R. solani diseases severity up to 73.2% and up to 62.7%, respectively.     

Sensitivity of Ascochyta rabiei populations to prothioconazole and thiabendazole

Ascochyta rabiei causes Ascochyta blight, a yield-limiting disease of chickpea (Cicer arietinum) world-wide. In 2007, fungal populations of A. rabiei resistant to the QoI group of fungicides were detected in the Northern Great Plains of the United States. Assays were conducted to determine fungal sensitivity for two alternative fungicidal modes of action. A total of 78 isolates of A. rabiei collected between 1983 and 2007 were screened to determine baseline sensitivity to the demethylation-inhibiting foliar fungicide, prothioconazole, and 100 isolates collected between 1987 and 2007 were screened for sensitivity to the methyl benzimidazole carbamate (MBC) fungicide, thiabendazole. Isolates were tested using an in vitro mycelial growth assay to determine the effective fungicide concentration at which 50% of fungal growth was inhibited (EC₅₀) for each isolate-fungicide combination. Baseline EC₅₀ values of prothioconazole ranged from 0.0526 to 0.2958 μg/ml, with a mean of 0.1783 μg/ml. Isolates of A. rabiei collected from 2007 to 2009 from North Dakota chickpea fields exposed to prothioconazole, were screened for prothioconazole sensitivity using the same assay. Mean EC₅₀ values for these isolates were 0.3544 μg/ml, 0.3746 μg/ml, and 0.7820 μg/ml, respectively. These values represent an approximate 2.0 (2007-2008) and 4.4-fold (2009) decrease in sensitivity from the baseline mean. EC₅₀ values of thiabendazole ranged from 1.192 to 3.819 μg/ml, with a mean of 2.459 μg/ml. No significant decrease in fungicide sensitivity was observed for thiabendazole. To date, no loss of Ascochyta blight control has been observed with the use of either prothioconazole or thiabendazole.     

Sensitivity of Sclerotinia sclerotiorum to fludioxonil: In vitro determination of baseline sensitivity and resistance risk

This work determined the sensitivity of field populations of Sclerotinia sclerotiorum (Lib.) de Bary before exposure to the fungicide fludioxonil (= baseline sensitivity) and assessed the risk of fludioxonil resistance. The mean EC₅₀ (Effective Concentration) and Minimum inhibitory concentration (MIC) values for fludioxonil based on inhibition of mycelial growth of 120 wild-type isolates were 0.015 ± 0.005 μg/ml and <0.05 μg/ml, respectively. Positive cross-resistance was not detected between fludioxonil and benzimidazole fungicides but was detected between fludioxonil and dicarboximide fungicides which are considered as high resistance risk fungicides by FRAC, even though these fungicides have different molecular structures. By growing wild-type isolates on potato dextrose agar (PDA) containing sublethal concentrations of the fungicide, we obtained four fludioxonil-resistant mutants with resistance factors (EC₅₀ resistant/EC₅₀ sensitive phenotypes) >2000. The laboratory fludioxonil mutants were less fitter than their parental isolates in terms of mycelial radial growth, pathogenicity and sclerotial production. Moreover, on PDA amended with NaCl, the laboratory fludioxonil mutants grew more slowly than their fludioxonil-sensitive parents, especially at lower concentrations of NaCl. According to the fitness of mutants and the cross-resistance between fludioxonil and dicarboximide fungicides, phenylpyrroles can be considered to pose a moderate resistance risk. In a field trial, fludioxonil provided greater control (over 90% disease control) of S. sclerotiorum than iprodione.     

Potential of extracts of the tropical plant Balanites aegyptiaca (L) Del. (Balanitaceae) to control the mealy bug, Maconellicoccus hirsutus (Homoptera: Pseudococcidae)

The effects of extracts of different parts of the perennial tropical plant Balanites aegyptiaca (L) Del., including various solvent extracts of roots, methanol extracts from leaves, fruits, flowers and roots, partially purified saponins obtained from its roots and a standard saponin were studied on the life cycle (adult longevity, number of eggs, crawlers, adults, weight of adults and % wax content) of a laboratory-reared parthenogenic line of the mealy bug, Maconellicoccus hirsutus (Homoptera: Pseudococcidae). Extracts derived from various parts of B. aegyptiaca (leaves, fruits, flowers, and roots in methanol) affected the life cycle of M. hirsutus with a methanol root extract being the most effective at a concentration of 500 μg ml⁻¹. Partially purified saponin of B. aegyptiaca and the commercial bark saponin extract (Sigma) from Quillaja saponaria at a concentration of 500 μg ml⁻¹ were effective in reducing the longevity of M. hirsutus. Significant reductions in oviposition by M. hirsutus were found for all the extracts at a concentration of 500 μg ml⁻¹. Extracts also affected the number of emerging crawlers, number of adults as well as the weight and wax content of emerging adults. These studies suggest that B. aegyptiaca plant extracts and saponins can be useful botanical insecticides for the protection of crops from mealy bugs.     

Insecticidal formulation based on Xylopia aethiopica essential oil and kaolinite clay for maize protection

Insecticidal formulations based on the essential oil of Xylopia aethiopica (Annonaceae) and kaolinite-clay (particles size less than 50 μm) are efficient in protecting stored grains. Ingestion-contact insecticide tests have been conducted using the maize weevil Sitophilus zeamais (Motsch.) (Curculionidae). Residual action of terpenic constituents adsorbed on clay was assessed. Bioassays indicated that S. zeamais adult mortality increased with the quantity of active ingredient. The formulation based on essential oil and clay produced mortality rates ranging from 22% at 2.5% (w/w) to 100% at 10% (w/w). The lethal concentration producing a 50% mortality rate (LC50) was 4.35% (w/w). Efficacy also depended on storage conditions and duration of the formulation, decreasing from 91 to 37% for formulation stored in closed boxes and from 91 to 17% for formulation stored in open boxes, after 1 and 15 days respectively.     

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.     

Biological control of Macrophomina phaseolina by chemotactic fluorescent Pseudomonas aeruginosa PN1 and its plant growth promotory activity in chir-pine

Ten strains of Pseudomonas aeruginosa (PN1 ˜ PN10) isolated from rhizosphere of chir-pine were tested for their plant growth promontory properties and antagonistic activities against Macrophomina phaseolina in vitro and in vivo. P. aeruginosa PN1 produced siderophore, IAA, cyanogen and solubilized phosphorus, besides producing chitinase and β-1,3-glucanase. In dual culture, P. aeruginosa PN1 caused 69% colony growth inhibition. However, cell free culture filtrate also posed inhibitory effect but to a lesser extent. After 90 days, P. aeruginosa PN1 increased plant growth and biomass in pots trial containing M. phaseolina-infested soil. PN1 showed the strong chemotaxis toward root exudates resulting in effective root colonization. Moreover, increased population in rhizosphere of these bacteria was also recorded after 90 days of treatment. Thus, chemotactic fluorescent P. aeruginosa PN1 exhibited strong antagonistic property against M. phaseolina, suppressed the disease and improved plant growth of the seedlings of chir-pine proving potential biocontrol agent.     

Berberine as a natural compound inhibits the development of brown rot fungus Monilinia fructicola

Peach brown rot is a severe disease caused by ascomycete fungus Monilinia fructicola and is primarily controlled by chemical fungicides. However, the utilization of fungicides has caused many problems such as microbial resistance, environmental pollution, and fruit contamination. The growing concern for food safety requires alternative management methods that are safe to humans and benign to the environment. Here we report that the extract from Coptis chinensis (a Chinese herb named “Huang Lian”) demonstrates a strong inhibition to M. fructicola. The 50% effective concentration (EC₅₀) of C. chinensis extract to M. fructicola was only 0.91 mg/mL, while the EC₅₀s to other fungi such as Botrytis cinerea and Alternaria solani were 14.09 mg/mL and 27.35 mg/mL, respectively. These results indicate that the extract of C. chinensis has a specific inhibition to M. fructicola. Subsequent partitioning of the ingredients in C. chinensis extract revealed that berberine, the conventional anti-bacterial alkaloid, was the predominant ingredient that exerted robust inhibition against M. fructicola. The EC₅₀ and minimum inhibitory concentration (MIC) against M. fructicola were as low as 4.5 μg/mL and 46.9 μg/mL, respectively. Compared to berberine’s analogs in C. chinensis such as palmatine, berberine tannate and jatrorrhizine, berberine showed the strongest inhibition against M. fructicola. This finding provided insight into the structure–activity relationship between berberine and its analogs. Notably, not only can berberine prevent spore germination and hyphal growth, it also inhibits the activity of cutinase secreted by M. fructicola, implying the potential function of berberine in reducing the pathogenesis of M. fructicola. In addition, the strong in vivo inhibition of berberine against M. fructicola was observed with no visual cytotoxicity noted to peach fruits, even at berberine concentration of 400 μg/mL. Note that this was much higher than its MIC value (46.90 μg/mL). Overall berberine, as a natural compound, may be a promising candidate in control of brown rot.     

Effects of crude seed extracts of Annona atemoya and Annona squamosa L. against the cabbage looper, Trichoplusia ni in the laboratory and greenhouse

Antifeedant, growth inhibitory and toxic effects of crude seed extracts of Annona squamosa and Annona atemoya from Fazenda Viveiro Bona, Parasisópolis – Minas Gerais, Brazil, were evaluated against the cabbage looper, Trichoplusia ni (Hübner) (Lepidoptera: Noctuidae) using different bioassays. Crude methanolic seed extracts deterred feeding of third instar T. ni larvae in a leaf disc choice bioassay. A. squamosa was ∼10 times more active as a feeding deterrent than A. atemoya (DC₅₀ = 2.3 mg/ml vs. 20.1 mg/ml). A. squamosa was ∼three times more active as a growth inhibitor than A. atemoya (EC₅₀ = 38.0 ppm vs. 117.0 ppm). Methanolic seed extracts of A. squamosa and A. atemoya were toxic to third instar T. ni larvae both through topical and oral application. A. squamosa was more toxic through feeding (LC₅₀ = 167.5 ppm vs. 382.4 ppm) whereas, A. atemoya exerted greater toxicity via topical application (LC₅₀ = 301.3 μg/larva vs. 197.7 μg/larva). Both A. squamosa and A. atemoya extracts reduced leaf area consumption and larval growth in a greenhouse experiment. Our results indicate that both A. squamosa and A. atemoya have potential for development as botanical insecticides, especially for local use in Brazil.     

Sensitivity to iprodione, difenoconazole and fludioxonil of Rhizoctonia cerealis isolates collected from wheat in China

Since the 1980s wheat sharp eyespot (WSE) caused mainly by Rhizoctonia cerealis has become one of serious diseases of wheat in China. In this study, the sensitivity of 89 R. cerealis isolates to different fungicides was evaluated using mycelial growth inhibition assays. The results showed that all R. cerealis isolates tested were sensitive to iprodione, difenoconazole and fludioxonil with mean EC₅₀ (effective concentration that results in 50% of mycelial growth inhibition) values of 0.419, 0.062 and 0.033 μg/ml, respectively. To evaluate the risk of fungicide resistance in R. cerealis, an attempt was made to induce resistant mutants in the laboratory. Although difenoconazole- and fludioxonil-resistant mutants were not obtained, we obtained seven independently iprodione-resistant (IR) mutants from 89 parental isolates. The EC₅₀ values for these IR mutants were greater than 100 μg/ml, whereas those for the original wild-type counterparts were less than 1 μg/ml. After having been subcultured on PDA for 10 generations, the IR mutants did not show any decrease in resistance to iprodione. Additionally, these IR mutants also showed resistance to fludioxonil but remained sensitive to difenoconazole. Osmotic sensitivity tests showed that the IR mutants were hypersensitive to osmotic stress generated by NaCl. Inoculation tests showed that all the IR mutants lost their ability to infect the host plant. Taken together, these results indicate that the current population of R. cerealis is sensitive to these fungicides and a fitness cost is associated with iprodione-resistant mutants of R. cerealis in both osmotic stress and pathogenicity. The information obtained in this study is useful in monitoring and managing fungicide resistance in R. cerealis populations in China.     

Phytotoxicity of Salvia spp. exudates

A study on thirteen Salvia species about the phytotoxicity of their aerial part exudates is reported. The selected species were: Salvia namaensis Schinz (1), Salvia fallax Fernald (2), Salvia disermas L. (3), Salvia chamaedryoides Cav. (4), Salvia confertiflora Pohl. (5), S. x jamensis J.Compton (6), Salvia buchananii Hedge (7), S. wagneriana Polak (8), Salvia scabra Linn.fil. (9), Salvia miniata Fernald (10), Salvia cacaliaefolia Benth.(11), Salvia adenophora Fernald (12), Salvia rutilans Carrière (13). For each species the inhibitory potential against Papaver rhoeas L. and Avena sativa L. was evidenced by dose-response studies and growth experiments performed both in Petri dish and in pot and also evaluated by means of germination indices and growth parameters. New indices useful for describing the phytotoxic response by a reduced number of testing solutions are proposed. Principal Component Analysis (PCA) of the germination data revealed that the exudate concentrations most significant in explaining the data variability were 1 and 2 μg/mL in Petri dish and 10 and 20 μg/mL in pot experiments. Root length and seedling weight were more important with respect to the other growth parameters.     

Effect of electrolyzed oxidizing water and continuous ozone exposure on the control of Penicillium digitatum on tangerine cv. ‘Sai Nam Pung’ during storage

The effect of electrolyzed oxidizing (EO) water in combination with ozone to control postharvest decay of tangerine cv. “Sai Num Pung” was investigated. The spore suspension containing 10⁵ conidia ml⁻¹ of Penicillium digitatum was prepared. EO water was generated by electrolysis of various concentrations of NaCl solution (5, 25, 50% and saturated NaCl). The spore suspension was inoculated into EO water and incubated at 27 °C for 1, 2, 4, 8 and 32 min. It was found that the EO water with saturated NaCl completely inhibited the spore germination of the fungus within 1 min. When the fruits inoculated with P. digitatum were washed in EO water at the same concentrations as previous experiment for 4, 8 and 16 min and stored at 5 °C for 18 days, it was found that immersion of the fruit in EO water for 8 min was the most effective to reduce disease incidences. Moreover, washing fruit in EO water and kept in a refrigerated chamber at 5 °C with continuous ozone exposure at a concentration of 200 mg l⁻¹ for 2 h day⁻¹ to extend storage life suppressed the disease incidence until 28 days. However, none of the treatments had any effect on the quality of fruit such as total soluble solids, titratable acidity, percent weight loss and peel color. Therefore EO water may be useful for surface sanitation and ozone has potential to control the recontamination of postharvest diseases in tangerine fruit in storage room.     

Insecticidal activities of crude extracts and phospholipids from Chenopodium ficifolium against melon and cotton aphid, Aphis gossypii

Several organic solvent extracts of Chenopodium ficifolium were tested for their insecticidal activity against melon and cotton aphid, Aphis gossypii, on cucumber plants. Both methanol and ethanol extracts, at 5000 μg ml⁻¹, were highly active giving over 80% control. The other crude extracts displayed moderate or weak insecticidal activity giving control in the range of 16–69%. Two phospholipids were isolated as insecticidal active substances from C. ficifolium. Their chemical structures were identified as 1-palmitoyl-2-(3-trans)-hexadecenoyl-sn-glycero-3-phosphoglycerol and 1-palmitoyl-2-linoleoyl-3-glycerophosphocholine by GC–MS, EDS, mass and NMR spectral analyses. Both compounds displayed a dose-dependent mortality of A. gossypii. Furthermore, the liquid formulation that was obtained by partitioning with n-hexane from the methanol extract of C. ficifolium controlled melon and cotton aphid on cucumber plants effectively. These results indicate that extracts of C. ficifolium have potential for development as botanical insecticides for controlling A. gossypii infesting cucumber plants.     

Efficacy of plant extracts and biocontrol agents against Pythium aphanidermatum inciting chilli damping-off

The fungitoxic effects of 66 medicinal plants belonging to different families were evaluated in vitro on Pythium aphanidermatum, the causal agent of chilli damping-off. Of these, Zimmu leaf extract (Allium sativum L. × Allium cepa L.) showed the highest inhibition of mycelial growth of P. aphanidermatum (13.7 mm). The antimicrobial compounds were isolated from Zimmu leaf extract and 22 compounds were identified through gas chromatography mass spectroscopy (GC–MS). Biocontrol agents Trichoderma viride and Pseudomonas fluorescens and Zimmu extract were also tested alone and together in vitro and in vivo experiments for control of P. aphanidermatum. The in vitro studies revealed that combination of T. viride + P. fluorescens + Zimmu leaf extract showed the highest mycelial growth inhibition over the control. Both antagonists were compatible with each other and with Zimmu leaf extract. The pot culture studies revealed that seed treatment with combined application of T. viride + P. fluorescens + Zimmu leaf extract was superior in reducing the pre and post-emergence damping-off incidence (8.3 and 17.0%, respectively), and increased the plant growth and yield (shoot length and root length of 13.7 and 6.3 cm, 146 g/plant, respectively) of chilli when compared to control.     

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.     

The use of adjuvants to improve spray deposition and Botrytis cinerea control on Chardonnay grapevine leaves

Spray adjuvants have the potential to improve deposition by effecting uniform distribution of the active ingredient on plant surfaces. In order to study whether such a qualitative improvement of spray deposition would lead to improved disease control, a laboratory experiment was conducted on artificially inoculated grapevine (cv. Chardonnay) leaves. Prior to inoculation with Botrytis cinerea conidia in a spore settling tower, leaves were sprayed to pre-runoff with 1 mL of a mixture of fenhexamid, a fluorescent pigment, and one of 15 selected commercial adjuvants to manipulate the deposition quality of a specific quantity of spray. Following an incubation period of 24 h at high relative humidity, leaf discs were plated onto Petri dishes with paraquat-amended water agar and rated for development of B. cinerea from isolated leaf discs 11 d later. Spray deposition on leaves was assessed with a spray assessment protocol using fluorometry, photomicrography and digital image analyses. B. cinerea incidences on the upper and lower surfaces of water-sprayed leaves averaged 90.4% and 95.8%, respectively. Despite full spray cover of leaves, applications with fenhexamid alone did not completely prevent infection and resulted in 34.6% and 40.8% B. cinerea incidences on the upper and lower surfaces of leaves, respectively. Through the addition of certain adjuvants, B. cinerea incidences were significantly reduced (incidences of 2.9–17.1% and 10.0–30.8%, respectively), while some adjuvants did not differ from the fungicide only treatment, even though they might have improved spray deposition. In a histopathology study using epifluorescence microscopy, distinct differences were observed in conidium mortality (20.5% vs. 31.2%), germination (60.4% vs. 51.4%) and germ tube lengths (27.8 μm vs. 19.7 μm) between Hydrosilicote and Solitaire in combination with fenhexamid, even though both adjuvants effected similar quantitative and qualitative spray deposition. The study clearly demonstrated the potential of adjuvants to improve the bio-efficacy of a fungicide directly through improved deposition on grapevine leaf surfaces, although bio-efficacy might be influenced by adjuvant mode of action in some cases.     

Efficacy and effects on yield of different fungicides for control of wet bubble disease of mushroom caused by the mycoparasite Mycogone perniciosa

Carbendazim, iprodione, prochloraz-Mn, thiabendazole and thiophanate-methyl were tested in vitro and in vivo for their effect on Mycogone perniciosa, the mycoparasite that causes wet bubble disease of white button mushroom. In vitro experiments showed that prochloraz-Mn (ED₅₀ = 0.006–0.064 μg ml⁻¹) and carbendazim (ED₅₀ = 0.031–0.097 μg ml⁻¹) were the most effective fungicides for inhibiting the mycelial growth of M. perniciosa, while iprodione (ED₅₀ = 1.90–3.80 μg ml⁻¹) was the least effective. The resistance factors calculated for the five fungicides were between 1.4 and 2. The results obtained suggest that there is very little risk that M. perniciosa will develop resistance to the fungicides assayed. The in vivo efficacy of fungicides for control of wet bubble was studied in two mushroom cropping experiments, which were artificially infected with two doses of M. perniciosa, 10⁶ and 10⁷ spores m⁻², respectively. There was, in the low dose inoculum experiment, a very high degree of effectiveness (96.5–100.0%) with all the fungicides assayed. However, iprodione performed poorly (20.5–24.4%) compared with the other fungicides (88.7–100.0%) in the high concentration inoculum experiment. The most effective treatments for controlling wet bubble did not improve the biological efficiency of Agaricus bisporus.