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The impact of combined environmental factors, such as temperature and CO₂, on the control of the powdery mildew of zucchini, caused by Podosphaera xanthii, and of different control measures has been studied on plants grown in phytotrons. Five experimental trials were conducted, and the powdery mildew severity of both treated and untreated zucchini plants was found to be significantly affected by the interaction between temperature (three different regimes: 16–18; 18–22; 22–26°C), CO₂ (two concentrations: 400–450 and 800–850 ppm) and the treatments. However, at the end of the trials, the efficacy of all the products was not affected by the different, tested environmental conditions. Sulphur consistently provided the highest disease control (75%–85% efficacy). Among the resistant inducers that were tested, calcium oxide was the most effective, in terms of powdery mildew control under all the conditions tested in phytotrons, reducing disease severity from 46% to 61%. Foliar applications of phosphite (14%–28% efficacy), Ampelomyces quisqualis (12%–23% efficacy) and potassium silicate (13%–24% efficacy) only slightly reduced the disease severity for all the tested temperature regimes and CO₂ concentrations, compared to the untreated control. The results obtained under our experimental conditions show that a possible increase in CO₂ concentration and temperature, which is expected for the next few years, should not influence the efficacy of the tested resistance inducers or of sulphur against powdery mildew on zucchini. Moreover, the suppressive effect of calcium oxide is in light of its possible use in greenhouses for zucchini powdery mildew control under 400–450 ppm of CO₂ and under enriched condition of 800–850 ppm of CO₂.

Background Aspergillus fumigatus, the causal agent of aspergillosis in humans, is commonly present as a saprophyte in various organic substrates, such as spoiled silages. Aspergillosis is generally combated with demethylation inhibitor (DMI) fungicides, but the recent appearance of resistant medical and environmental strains made current treatment strategies less reliable. The goal of this study was to determine the evolution of A. fumigatus populations during the ensiling process of whole-crop corn, high moisture corn and wet grain corn, and to monitor the sensitivity of isolates from treated and untreated fields to one medical and one agricultural DMI fungicide. Results A. fumigatus was isolated from fresh forage at harvest at rather low concentrations (102 cfu g-1 ). The low frequency lingered during the silage process (at 60 and 160 days), whereas it significantly increased during air exposure (at 7 and 14 days of air exposure). Field treatment of corn with a mixture of prothioconazole and tebuconazole did not affect the sensitivity of A. fumigatus isolates. One of 29 isolates from the untreated plot was resistant to voriconazole. A unique amino acid substitution (E427K) was detected in the cyp51A gene of 10 of 12 sequenced isolates, but was not associated with DMI resistance. Conclusion A. fumigatus significantly increased during aerobic deterioration of ensilaged corn after silo opening, compared with the low presence in fresh corn and during ensiling. Field treatment of corn with DMI fungicides did not affect the sensitivity of A. fumigatus isolates collected from fresh and ensiled corn. © 2019 Society of Chemical Industry.

Some plant species are capable of significant reduction of xylem embolism during recovery from drought despite stem water potential remains negative. However, the functional biology underlying this process is elusive. We subjected poplar trees to drought stress followed by a period of recovery. Water potential, hydraulic conductivity, gas exchange, xylem sap pH, and carbohydrate content in sap and woody stems were monitored in combination with an analysis of carbohydrate metabolism, enzyme activity, and expression of genes involved in sugar metabolic and transport pathways. Drought resulted in an alteration of differential partitioning between starch and soluble sugars. Upon stress, an increase in the starch degradation rate and the overexpression of sugar symporter genes promoted the efflux of disaccharides (mostly maltose and sucrose) to the apoplast. In turn, the efflux activity of the sugar-proton cotransporters caused a drop in xylem pH. The newly acidic environment induced the activity of apoplastic invertases leading to the accumulation of monosaccharides in the apoplast, thus providing the main osmoticum necessary for recovery. During drought and recovery, a complex network of coordinated molecular and biochemical signals was activated at the interface between xylem and parenchyma cells that appeared to prime the xylem for hydraulic recovery.

BACKGROUND Pythium species attack various vegetable crops causing seed, stem and root rot, and 'damping-off' after germination. Pythium diseases are prevalently controlled by two classes of fungicides, QoIs with azoxystrobin and phenlyamides with mefenoxam as representatives. The present study aimed to test the sensitivity of six Pythium species from different vegetable crops to azoxystrobin and mefenoxam and differentiating species based on ITS, cytochrome b and RNA polymerase I gene sequences. RESULTS The inter- and intra-species sensitivity to azoxystrobin was found to be stable, with the exception of one Pythium paroecandrum isolate, which showed reduced sensitivity and two cytochrome b amino acid changes. For mefenoxam, the inter-species sensitivity was quite variable and many resistant isolates were found in all six Pythium species, but no RNA polymerase I amino acid changes were observed in them. ITS and cytochrome b phylogenetic analyses permitted a clear separation of Pythium species corresponding to globose- and filamentous-sporangia clusters. CONCLUSION The results document the necessity of well-defined chemical control strategies adapted to different Pythium species. Since the intrinsic activity of azoxystrobin among species was stable and no resistant isolates were found, it may be applied without species differentiation, provided it is used preventatively to also control highly aggressive isolates. For a reliable use of mefenoxam, precise identification and sensitivity tests of Pythium species are crucial because its intrinsic activity is variable and resistant isolates may exist. Appropriate mixtures and/or alternation of products may help to further delay resistance development. © 2018 Society of Chemical Industry.

BACKGROUND Aspergillus fumigatus is a widespread fungus that colonizes dead organic substrates but it can also cause fatal human diseases. Aspergilloses are treated with demethylation inhibitor (DMI) fungicides; however, resistant isolates appeared recently in the medical but also environmental area. The present study aims at molecular characterizing and quantifying A. fumigatus in major environmental habitats and determining its sensitivity to medical and agricultural DMI fungicides. RESULTS A. fumigatus was isolated only rarely from soil and meadow/forest organic matter but high concentrations (103 to 107 cfu/g) were detected in substrates subjected to elevated temperatures, such as compost and silage. High genetic diversity of A. fumigatus from compost was found based on SSR markers, distinguishing among fungal isolates even when coming from the same substrate sample, while subclustering was observed based on mutations in cyp51A gene. Several cyp51A amino acid substitutions were found in 15 isolates, although all isolates were fully sensitive to the tested DMI fungicides, with exception of one isolate in combination with one fungicide. CONCLUSION This study suggests that the tested A. fumigatus isolates collected in Italy, Spain and Hungary from the fungus’ major living habitats (compost) and commercial growing substrates are not potential carriers for DMI resistance in the environment.

Seed-dressing with essential oils from aromatic plants (savoury and thyme) and with antagonistic bacteria strains (Pseudomonas spp.) was tested after a hot water treatment against Alternaria radicina inoculated on carrot seeds. Seed treatments by immersion in water at 55°C for 10 min improved the efficacy on pathogen control of the treatments with biocontrol agent strains more than the efficacy of those with essential oils. Pretreatment with hot water also increased the germination rate of carrot seeds. A mild phytotoxic effect was observed on the germination rate and the fresh biomass obtained from seeds treated with both essential oils. Treatments with essential oils and antagonistic bacteria presented positive results when combined with hot water dipping of seeds on control of A. radicina, but the effect was not completely additive. The formulation and the application method of the non-chemical products tested are critical on their development as an alternative strategy on seed disinfection.

The pathogenicity of different isolates of Fusarium oxysporum obtained from plants of Gerbera (Gerbera jamesonii), Chrysanthemum (Chrysanthemum morifolium), Paris daisy (Argyranthemum frutescens) and African daisy (Osteospermum sp.), all in the family Asteraceae, was tested on different cultivars of these hosts, to assess their pathogenicity. The reactions were compared with those of isolates of F. oxysporum f. sp. chrysanthemi and of f.sp. tracheiphilum obtained from the American Type Culture Collection. We found that isolates of F. oxysporum f. sp. chrysanthemi can be distinguished as three physiological races on the basis of their pathogenicity to the panel of differential cultivars. Sequencing of the intergenic spacer (IGS) region of ribosomal DNA (rDNA) and phylogenetic analysis showed that the Fusarium races fell into three phylogenetic groups, which coincided with those observed in pathogenicity tests. Analysis of the IGS sequences revealed a high degree of similarity among strains from Italy and Spain from different host species, suggesting that recent outbreaks in these ornamentals were probably caused by introduction of infected nursery material from a common origin.

Flavescence doree (FD) is among the major grapevine diseases causing high management costs; curative methods against FD are unavailable. In FD-infected plants, decrease in photosynthesis is usually recorded, but deregulation in stomatal control of leaf gas exchange during FD infection and recovery is unknown. We measured the seasonal time course of gas exchange rates in two cultivars (‘Barbera’ and ‘Nebbiolo’) during the term of 1 year when grapevines experienced a water stress and another with no drought, with difference in gas exchange rates in response to FD infection and recovery as assessed by symptom observation and phytoplasma detection through PCR analysis. Chlorophyll fluorescence was also evaluated at the time of maximum symptom severity in ‘Barbera’, the cultivar showing the most severe stress response to FD infection, causing the highest damage in vineyards of north-western Italy. In FD-infected plants, net photosynthesis and transpiration gradually decreased during the season, more during the no drought year than during drought. During recovery, healthy (PCR negative) plants infected 2 years before, but not those infected an year before, regained the gas exchange performances to the level as measured before infection. The relationships between stomatal conductance and the residual leaf intercellular CO2 concentration (ci) discriminated healthy versus FD-infected and recovered plants; at the same ci, FD-infected leaves had higher non-photochemical quenching than healthy ones. We conclude that metabolic, not stomatal, leaf gas exchange limitation in FD-infected and recovered grapevines is the basis of plant response to FD disease. In addition, we also suggest that such response is dependent upon water stress, by showing that water stress superimposes on FD infection in terms of stomatal and metabolic non-stomatal limitations to carbon assimilation.

The aim of this study was to evaluate the effect of potassium silicate administration and of electrical conductivity of nutrient solution in three experiments against Colletotrichum gloeosporioides infection on basil (Ocimum basilicum L. cv Genovese Gigante) grown in a closed soilless system. Potassium silicate was added at 100 mg/l of nutrient solution at three different levels of electrical conductivity: 1.5–1.6 mS/cm (E.C.1), 3–3.2 mS/cm (E.C.2, 0.70 g/l NaCl) and 4–4.2 mS/cm (E.C.3, 0.95 g/l NaCl). Basil plants were inoculated with C. gloeosporioides spores 21–31 days after sowing or placing the pots on the channels, applying 5 ml of conidial suspension to each treatment. The increased electrical conductivity of the nutrient solution generally reduced the incidence and severity of the disease, with the highest electrical conductivity (E.C.3) providing the best results. The addition of potassium silicate to the different nutrient solutions showed a significant reduction in both incidence and severity of the disease compared to a solution without silicate and the best results were given by the addition of silicate with the highest electrical conductivity (E.C.3) in all the trials carried out. The combination of high electrical conductivity and potassium silicate supplied gave good results. The possibility and benefits of applying Si amendments in practice are examined.

Between 2006 and 2008, 146 isolates of Fusarium spp. were obtained from bakanae-diseased rice plants and seeds from the major rice-growing regions of Italy. These isolates were identified based on translation elongation factor (EF-1a) sequence and pathogenicity tests were used to assess their aggressiveness against the susceptible rice cultivar Galileo. Use of the EF-1a sequence gave reliable identification and showed that Fusarium fujikuroi, the causal agent of bakanae disease, was the most abundant Fusarium spp. isolated. These data were confirmed by inoculation of the isolates to rice seeds which were then germinated in the greenhouse, showing that only F. fujikuroi isolates were able to cause bakanae disease. Pathogenic isolates were identified with different levels of aggressiveness. Phylogenetic analysis based on EF-1a sequences generated a tree which separated the various Fusarium species into different clusters with high bootstrap values.

The efficacy of plant essential oils was evaluated on apples, cv. Golden Delicious, cv. Granny Smith, cv. Red Chief and cv. Royal Gala, at postharvest to control Botrytis cinerea and Penicillium expansum. The essential oils from basil (Ocimum basilicum), fennel (Foeniculum sativum), lavender (Lavandula officinalis), marjoram (Origanum majorana), oregano (Origanum vulgare), peppermint (Mentha piperita), rosemary (Rosmarinus officinalis), sage (Salvia officinalis), savory (Satureja montana), thyme (Thymus vulgaris) and wild mint (Mentha arvensis) were tested at different concentrations. All the essential oils were from mediterranean plants. The fruits were artificially wounded and inoculated with a suspension at 1 × 105 conidia/ml of each pathogen. After 12 h, emulsions at 1% and 10% of each essential oil were dropped into each inoculated wound. A tebuconazole chemical control and an inoculated control were also included. All the treated fruit were stored at 4 ± 1°C. After 15 and 30 days, the diameter of the rot around each wound was measured. The results revealed that the efficacies of the essential oils tested were cultivar- and storage time-dependent. In any case, treatments with essential oils from oregano, savory and thyme showed significant efficacy in all apple cultivars tested. Treatments with essential oil emulsions at 10% were phytotoxic for all the apple cultivars evaluated. Copyright © 2010 John Wiley & Sons, Ltd.

A total of 35 isolates of Fusarium oxysporum f.sp. eustomae obtained from diseased Eustoma grandiftorum plants in northern Italy, showing typical Fusarium wilt symptoms, were analysed for their genetic variability and molecular identification. Genetic diversity of the isolates was studied by using random amplified polymorphic DNA (RAPD). This analysis clustered the isolates into three groups at a genetic similarity of 69%. Sequence analysis of RAPD fragments led to the design of a pair of specific primers that amplify a 505-bp SCAR (sequence characterized amplified region) marker (SCAR 505 ) which was used to rapidly detect F. oxysporum f.sp. eustomae on Eustoma grandiflorum plants. In a temperature-controlled chamber, detection of the pathogen by PCR was 100% successful in root and stem samples of infected but still symptomless plants. The diagnostic procedure could be completed in 1 day and allowed rapid and reliable detection of the pathogen in asymptomatic plants in the early stages of disease development.

The random amplified polymorphic DNA (RAPD) technique was used to analyze the total genomic DNA of pathogenic isolates of Fusarium oxysporum on Gerbera jamesonii by comparing them to representatives of the formae speciales chrysanthemi and tracheiphilum. A close genetic relationship was observed among most of the new isolates from G. jamesonii. They shared RAPD markers with the tested representatives of the forma specialis chrysanthemi. Some isolates of those tested from diseased G. jamesonii were placed in a different cluster, which included representative isolates of forma specialis tracheiphilum. This is the first report of F. oxysporum f.sp. tracheiphilum on G. jamesonii. A rapid protocol for DNA extraction directly from fungal colonies grown on potato dextrose agar allowed complete analysis in less than 4 h.

In this work, a protocol for zoospores production of Phytophthora cryptogea, an economically important plant pathogen was optimized. Five different concentrations of zoospores (5 × 10 5 , 5 × 10 4 , 5 × 10 3 , 5 × 10 2 , 5 × 10 1 zoospores/ml) from four different isolates of P. cryptogea (Maria 1, Maria 2, S3 1-A, Amazzone) were used as inoculum on pot marigold (Calendula officinalis) and gerbera (Gerbera jamesonii) plants. Maria 1 was the most virulent isolate both on pot marigold and gerbera plants according to disease severity. A rapid and sensitive pathogen DNA extraction protocol suitable for large quantities of plant samples was adopted. Conventional polymerase chain reaction (PCR) was able to detect the pathogen in artificially inoculated symptomless pot marigold (collected day 12) and gerbera plants (day 8) after pathogen inoculation, with the suspension of 5 ×10 5 , 5 ×10 4 , 5 ×10 3 P. cryptogea zoospores/ml. Real-time PCR showed the possibility to detect the pathogen in artificially inoculated symptomless pot marigold (collected day 8) and gerbera plants (day 4) after pathogen inoculation, with the suspension of 5 × 10 5 , 5 × 10 4 P. cryptogea zoospores/ml. The first symptoms appeared on pot marigold plants 14 days after pathogen inoculation and on gerbera plants 10 days after inoculation. Real-time PCR showed the possibility to detect the pathogen 4 days before conventional PCR and 6 days before the appearance of disease symptoms both on pot marigold and gerbera plants.

In the present article we have ascertained the presence of a consortium of ectosymbiotic bacteria belonging to Serratia, Achromobacter, Bacillus and Stenotrophomonas genera associated to the mycelium of the antagonistic Fusarium oxysporum MSA 35 [wild-type (WT) strain]. Morphological characterization carried out on the WT strain, on the F. oxysporum MSA 35 without ectosymbionts [cured (CU) strain] and on the pathogenic F. oxysporum f.sp. lactucae (Fuslat 10) showed that the ectosymbionts, present only in the WT strain, caused a depleted production of micro conidia and aerial hyphae, and a change in shape and dimension of the latter. Virulence tests showed that the cured Fusarium was a pathogenic strain and, as shown by polymerase chain reaction and microscope analysis, pathogenicity was correlated with the capability of the cured hyphae of penetrating lettuce roots. Accordingly, the hyphae of the WT strain were impaired in entering the plant roots. Typing experiments provided evidence that both CU and WT strains belong to F. oxysporum f.sp. lactucae. This implies that the antagonistic effect of WT Fusarium is not a fungal trait, but it is due to the interaction with the ectosymbiotic bacteria. Expression analysis showed that fmk1, chsV and pl1 genes involved in F. oxysporum pathogenicity are not expressed in the WT strain whereas they are expressed in the cured fungus. These results, together with the hyphal characteristics, suggest that the inability of WT strain to penetrate the plant roots could be due to alterations in the expression profile of cell wall-degrading enzymes. In conclusion, we demonstrated a modulation of F. oxysporum gene expression in response to the interaction with the ectosymbiotic bacteria. Preliminary researches indicated that the presence of bacteria attached to the hyphae of antagonistic F. oxysporum is not an isolated phenomenon. Further investigations are necessary to better understand the rule and the diffusion of ectosymbiotic bacteria among antagonistic Fusarium.

The pathogenicity of five isolates of Fusarium oxysporum obtained from infected gerbera (Gerbera jamesonii), chrysanthemum (Chrysanthemum morifolium), Paris daisy (Argyranthemum frutescens) and African daisy (Osteospermum sp.) plants was tested on some varieties of the following Compositae hosts: C. morifolium, G. jamesonii, Argyranthemum frutescens (Paris daisy) and Osteospermum sp. and compared with the host range and pathogenicity of an isolate of F. oxysporum f. sp. chrysanthemi obtained from the ATCC collection. The results indicated that isolates of F. oxysporum from G. jamesonii as well as those from A. frutescens and Osteospermum sp. belong to the forma specialis chrysanthemi. The isolate from gerbera was virulent on all tested varieties of gerbera, C. morifolium, A. frutescens and Osteospermumsp. Similar results were obtained testing the isolates obtained from A. frutescens and Osteospermumsp. The strain from C. morifolium infected cultivar of gerbera, A. frutescens and Osteospermum sp. The pathogenicity of isolate of F. oxysporum f. sp. chrysanthemi obtained from the ATCC showed a different cultivar range particularly in the case of chrysanthemum and gerbera.

From 2002 to 2004, wilted plants of different species of rocket (Eruca vesicaria and Diplotaxis spp.) were found for the first time in Europe, in greenhouse cultivations in Piedmont and Lombardy, northern Italy. The causal agent of the disease was found to be Fusarium oxysporum. Vegetative compatibility analysis was carried out on 46 isolates of the fungus, 41 of them obtained from wilted rocket (E. vesicaria and D. tenuifolia) and five reference strains, in order to increase the knowledge on the causal agent of recent epidemics of Fusarium wilt on rocket in Italy. The analysis showed the presence of two vegetative compatibility groups (VCGs) (VCG 0101 and VCG 0220) pathogenic on both kinds of rocket. The two VCG populations, which were classified as formae speciales conglutinans and raphani, respectively, are spread in the area of epidemics but are not related to the host species from which they were isolated (D. tenuifolia or E. vesicaria). This finding shows the heterogeneity of the causal agent of Fusarium wilt on rocket in Italy.

A real-time polymerase chain reaction (PCR) assay using a TaqManprobe was developed to detect the causal agent of wilt and crown rot of basil from infec- ted plants and seed in Italy. The aim of the study was to diminish testing time, previously performed using nested-PCR, and to create the conditions for future automation. The sensitivity of the assay was shown to be similar to the detection limit of the available nes- ted-PCR procedure. The advantages of real-time PCR system include halving of the testing time, as well as the ability to identify both internally and externally infected seed to the sensitivity of 1 pg of genomic DNA. The assay was able to detect the presence of the pathogen in infected seed up to a sensitivity of 24 (SD: ±10) CFU per 100 seeds.

The random amplified polymorphic DNA (RAPD) technique was used to analyse total genomic DNA of 10 isolates of a new Fusarium oxysporum pathogenic on Argyranthemum frutescens (Paris daisy), by comparing them with representatives of the formae speciales basilici, chrysanthemi, cyclaminis, dianthi, gladioli, lilii, lycopersici, melonis, pisi, radicis-lycopersici, tracheiphilum, and a non-pathogenic isolate of F. oxysporum. A close genetic relatedness was observed among most of the new isolates from A. frutescens. These isolates also shared RAPD markers with the tested representatives of the forma specialis chrysanthemi. A single isolate among those tested from diseased A. frutescens was placed in a different cluster, which included representative isolates of forma specialis tracheiphilum. All the new isolates from A. frutescens, with the exception of the single divergent one, could be identified by their characteristic amplification profile, using selected random primers. A rapid protocol for DNA extraction directly from fungal colonies grown on Fusarium selective medium allowed the complete analysis in less than 4 h.

The stability of a genetically modified strain of Fusarium oxysporum used as antagonist against phytopathogenic formae speciales of F. oxysporum was evaluated both in vitro and in microcosm assays. The Escherichia coli hygromycin B phosphotransferase gene (hph), conferring hygromycin B resistance, was introduced by genetic transformation into a recipient strain marked by benomyl resistance and a dark red pigmentation. Hybridization with the complete plasmid suggested that the integration had generally occurred in a multiple-tandem array at multiple sites. Among nine independent transformants tested, only three of them were mitotically stable after four rounds of vegetative growth with no selective pressure, while six showed various changes in the integration pattern. One transformant had lost the ability to grow in the presence of hygromycin B. In soil microcosms all the transformants maintained the hygromycin B resistant phenotype, but six of them showed rearrangement of transforming DNA. Only one strain (coded T26.40) underwent no obvious rearrangement both after in vitro growth and after recovery from the soil microcosm. The nine transformants were used in three biological control experiments against Fusarium wilt of carnation in comparison to two untransformed reference strains and to the recipient mutant. A high degree of variability in the biocontrol activity was observed throughout the experiments and only transformant T26.40 consistently controlled the incidence of disease. The results are discussed in relation to risk assessment of the release of transgenic antagonistic fungi.

A propiconazole-resistant Trichoderma harzianum strain with high phylloplane survival capability was transformed with the E. coli hygromycin B phosphotransferase gene (hph), coding for hygromycin B resistance. Four transformants were analysed for survival ability on the phylloplane of tomato plants grown under glasshouse conditions in comparison with their prototype and a yellow, hygromycin B-sensitive mutant. Over 2 weeks, the four transformants showed higher survival rates in comparison with the wild-type strain. The yellow mutant TF3/973 did not significantly differ in survival from the transformants. Both hygromycin B resistance and mitotic stability of transformants were evaluated during growth in vitro and after reisolation from tomato phylloplane. Hybridization patterns with the complete plasmid indicated that all four transformants were mitotically stable after several rounds of vegetative growth without selective pressure and during 2 weeks on tomato plants. None of the transformants had lost the ability to grow in the presence of both propiconazole and hygromycin B after growth under the same conditions. The results are discussed in relation to risk assessment of the release of transgenic fungi.

Results are reported for four experimental trials carried out against Botrytis cinerea on wine grapes in Piedmont (Northern Italy) during 1979 and 1980, using fungicides with different mechanisms of action, alternately or in mixture. Good effectiveness was obtained not only with the exclusive use of dicarboximide fungicides but also with alternated spray programmes, for example using benomyl or captafol or dichlofluanid for the first two sprays and the dicarboximides only for the last two sprays. It was possible to reduce the number of sprays with the dicarboximides to only one (at beginning of ripening), by, for example, alternating dichlofluanid, vinclozolin and benomyl. Good results were also obtained using half-rate mixtures of fungicides with different mechanisms of action, for example benomyl or dichlofluanid mixed with one of the dicarboximides. In this way the selection pressure exercised by the dicarboximides is decreased. Moreover, the cost of treatments is reduced owing to the lower price of benomyl, dichlofluanid and captafol and because these fungicides are effective not only against B. cinerea but also against powdery mildew (benomyl) or downy mildew (dichlofluanid and captafol).