Fungicide residues in pears, apples and grapes after post harvest treatments by thermonebulization

In the context of trials conducted in cold store, in order to investigate efficacy of fruit protection applying agrochemicals, the fate during the storage of two active ingredients (a.i.): pyrimethanil and imazalil, have been studied. The experimental trials were done on eight varieties of apples and pears and two of grapes. Only field use of named pesticides was already authorised on fruits and maximum residue limits (MRL) fixed. In the present biennial experimentation (apples and pears in the first year; grapes in the second one). Two fungicides formulations were applied and treatments have been executed by thermonebulization technique. The storage cell, had a volume of 110 m3 filled up with the fruits until 80% of its capacity. Each fruit variety constituted one thesis of 4 replicates. For each trial, only one treatment was performed at the beginning of conservation period (72 days for pears and apples, and 57 days for grapes). In order to estimate fungicides persistence, residues determinations have been carried out. From results obtained especially on grape, not homogenous residues distribution on treated commodities was observed. For all theses, residue level has not evidenced statistically significant decrement as function of time, behaviour not as observed in field tests. The following average values of residues have been determined: in apples and pears, 0.7 mg/kg of imazalil much lower than 5 mg/kg (RML fixed for field use), and 1.2 mg/kg of pyrimethanil little higher than 1 mg/kg (RML fixed for field use); in grapes 0.5 mg/kg pyrimethanil much lower than 3 mg/kg (RML fixed for field use).     

Sterol demethylation inhibitor fungicides as disruptors of insect development and inducers of glutathione S-transferase activities in Mamestra brassicae

To study physiological and biochemical effects of demethylation inhibitor (DMI) fungicides on non-target insects, larvae of the cabbage moth, Mamestra brassicae L., were exposed orally to propiconazole, (R,S)-1-[2-(2,4-diclophenyl)-4-propyl-1,3-dioolan-2-ylmetyl]-1H-1,2,4-triazole (100, 200 and 600 mg L(-1)) and fenpropimorph, (+/-)-cis-4-[3-(4-tert-butylphenyl)-2-methylpropyl] 2,6-dimethylmorpholinc (10, 100, 200 and 600 mg L(-1)) in a semi-synthetic diet. Ten mg L(-1) of fenpropimorph reduced larval weight and induced in vitro glutathione S-transferase activity. Reduced larval and pupal growth rate, reduced survival, prolonged developmental time, and altered patterns of larval survival and adult emergence were found for one or both fungicides in at least one of the concentrations tested. The results suggest, that although the use of agricultural fungicides is generally regarded as of minor ecotoxicological consequence for insects, feeding on DMI-treated crops may influence insect fitness, and may also leave them susceptible to pesticide treatments or to residues of pesticides and other pollutants in their food. Standard methods to detect such effects should be developed for use in the environmental risk assessment of these products.     

Screening preharvest/postharvest strategies to prevent fruit rot decay

In fruit growing preharvest sprayings in the orchard are mainly applied to protect fruit from decaying. Next to multisite fungicides (captan, thiram, tolylfluanid) the most commonly used products recognized for the Belgium market are Bellis (pyraclostrobin & boscalid) and the combination of Topsin M (thiophanate-methyl) and Frugico (diethofencarb). In general the spraying schedule varies depending on weather conditions (infection risk), preharvest interval of available fungicides, fruitgrower and cultivar of pome fruit (apple/pear). Facing the climatological conditions before picking the residue loading on the fruit surface can differ enormously. Also wet (pre)grading is considered to decrease the product residue resulting to fruits which are less protected before entering the cold storage room. In this context a partially replacement of the preharvest treatments by one postharvest application could offer a reliable alternative to the PPP reduction program (Plant Protection Products) in the orchard. A standardized application method by dipping or drenching will cover the fruits homogenically resulting in a rationalized fungicide use compared to the preharvest sprayings in the orchard. For the Belgium market Philabuster (imazalil & pyrimethanil) is registered for postharvest treatments since for this product a proper solution for the waste water of postharvest uses was developed to protect surface waters (Funds technology). Philabuster provides an advanced mould control towards fruit rot pathogens Gloeosporium spp., Botrytis cinerea and Penicillium spp. In this context several trials were set up to evaluate the biological efficacy of Philabuster alone or in combination with preharvest sprayings in the orchard. In concrete different preharvest spraying schedules were applied in the last six weeks before harvest on apple and pear facing parameters as rational fungicide use, antifungal effectiveness and cost price. The purpose was to select the optimal combination in use of preharvest fungicides with Philabuster as postharvest treatment, which offer full protection towards all key pathogens in apple and pear.     

Antifungal action of chitosan in combination with fungicides in vitro and chitosan conjugate with gallic acid on tomatoes against Botrytis cinerea

In the present work, a positive effect was obtained by using low molecular weight chitosan compounds in combination with synthetic fungicides. Antifungal activity against Botrytis cinerea, determined by the radial growth method, was more than 75%, with a 25?×?10^??10 g/L concentration of fludioxonil or difenoconazole in compounds. Metabolic activity of B. cinerea fungus was about 15% when using a chitosan compound containing fludioxonil at a concentration of 25?×?10^??7 g/L. The combined action of chitosan with difenoconazole at a fungicide concentration of 25?×?10^??4 g/L is 2–3 times more effective than the action of each component separately. Results of studies for artificially inoculated B. cinerea tomato fruit when treated with low molecular chitosan and chitosan conjugate with gallic acid reduced the frequency of rotting fruit by 50 and 83%, respectively. Chitosan-gallic acid conjugate were obtained from chitosans with Mw of 28 kDa (Ch28GA) was proved to be effective as a preventive treatment for 3 days and can potentially be used as a biofungicide against B. cinerea on tomatoes in the post-harvest period.

     

艾叶超临界CO2萃取物的抑菌活性研究

本文研究了艾叶超临界CO2萃取物的抑菌活性。结果表明:艾叶超临界CO2萃取物对细菌、酵母、霉菌都有一定的抑菌作用,在酸性环境和碱性环境较为显著,对大多数细菌、酵母、霉菌的MIC不超过0.78 g/L。高温长时间处理对萃取物抑菌活性影响较大,但高温瞬时或低温处理对萃取物抑菌活性影响不大。     

一株祼脚菇属菌株产抗菌活性物的基础液体培养碳、氮源等优化及其对柑橘青绿霉菌的作用

江西是柑橘类水果的主要产区之一,青绿霉菌是导致柑橘采后腐烂的主要病害,化学杀菌剂可产生病原抗药性和残留,一株具有拮抗此病菌的祼脚菇属菌株0612-9被发现。为了探索其产活性物质的代谢培养和活性作用,采用菌丝生长速率法和显微观察法测定了其抗菌活性,单因素试验、Plackett-Burman、最陡爬坡试验和Box-Behnken试验响应面设计等优化出液体培养基组成和培养条件。结果显示:液体培养基组成和培养条件为:马铃薯279.94g/L,葡萄糖30g/L,麸皮8.87g/L,MgSO₄·7H₂O 0.47g/L,KH₂PO₄ 2g/L,发酵周期10d,接种量5 ml/100 ml,转速180 r/min,摇床温度26℃,装液量100ml/250ml,发酵液抗病菌活性从42.26%提高到55.77%。对意大利青霉和指状青霉的EC₅₀分别为69.25g/L和56.70g/L,最小杀菌浓度250μg/ml低于阳性对照物抑霉唑的500μg/ml,并且对病原菌菌丝体有一定的毒害作用。该祼脚属菌株有潜在开发成柑橘采后保鲜剂的价值。     

外源抗坏血酸处理对人工脱涩柿果的保鲜作用

该研究以广西中部地区的早熟品种‘牛心柿’(Diospyros kaki cv.Niuxin)的果实为材料,研究果实萼片施用5%抗坏血酸溶液处理对CO_2脱涩柿果采后贮藏期果实软化和细胞壁降解以及抗氧化体系等相关生理生化指标的影响。结果表明:抗坏血酸对人工脱涩后‘牛心柿’果实的保鲜效果明显,与空白和去离子水对照相比,外源抗坏血酸处理更好地延缓柿果实硬度的降低和总色差的升高。抗坏血酸处理后7~15 d,原果胶的下降速率和可溶性果胶的上升速率得以延缓,细胞壁降解酶PG和Cx活性均低于空白和对照。同时,外源抗坏血酸阻滞了整个贮藏期果实内源抗坏血酸的减少,并抑制了POD的活性和MDA的积累,果实自身抗氧化体系功能受到保护。此外,抗坏血酸处理还在一定程度上提高了果实还原糖的含量。因此,采后外源施用抗坏血酸不仅可以延长脱涩后鲜食柿果的货架期,而且还有助于提高果实的商品品质。     

Integrated control of Colletotrichum gloeosporioides on mango fruit in Taiwan by the combination of Bacillus subtilis and fruit bagging

Anthracnose disease caused by Colletotrichum gloeosporioides in Jingkwang mango grown in Taiwan was significantly reduced by the integration of fruit bagging with either B. subtilis strain LB5 or fungicides. The combined treatments were most effective in reducing early infection during the 2004 season, leading to 56.4 and 58.3% reduction, respectively, while in 2003 reduction accounted for 51 and 52.3%, respectively. Post-harvest application of B. subtilis strain LB5 cell suspensions on fruits already treated by bagging, bagging+LB5 and baggingfungicides in the field reduced anthracnose incidence significantly at all tested concentrations. These results indicate that biocontrol efficacy of B. subtilis LB5 may be due to the prevention of early fruit infection, thereby reducing significantly anthracnose incidence in ripening fruits to much lower levels than those obtained by using a conventional single post-harvest treatment.     

Assessment of Sensitivities to Anilinopyrimidine- and Strobilurin-fungicides in Populations of the Apple Scab Fungus Venturia inaequalis

The sensitivity of Venturia inaequalis populations to the anilinopyrimidine fungicides pyrimethanil and cyprodinil was analysed by microscopic in vivo analysis of conidiophore formation. The sensitivity to the strobilurin kresoxim-methyl was analysed using an in vitro germination assay and by determination of the diseased leaf area and conidia produced in vivo. Baseline sensitivities were determined with V. inaequalis populations from control orchards that had never been treated with fungicides. Comparison of the baseline sensitivities with sensitivities of populations obtained from orchards that had received 43 anilinopyrimidine treatments over 4 years, or from an orchard with 54 kresoxim-methyl treatments over 6 years indicated that no resistance to these fungicides has developed at the sites sampled.     

Expression and homology modeling of sterol 14alpha-demethylase from Penicillium digitatum

Green mold of citrus, caused by Penicillium digitatum, is the most serious postharvest disease of citrus. Sterol 14alpha-demethylase (CYP51) is one of the key enzymes of sterol biosynthesis in biological kingdoms and is a prime target of antifungal drugs. To exploit novel 14alpha-demethylase inhibitor (DMI) fungicides, DNA and total RNA were isolated from P. digitatum. The CYP51 of P. digitatum was cloned and expressed in Escherichia coli, yielding recombinant protein with a molecular weight of c. 59 kDa. The P. digitatum CYP51 protein (PdCYP51) was purified and polyclonal antibodies were prepared. Compared with the sequence of P. digitatum PD5 in GenBank, there were four mutated nucleotides which resulted in four mutated amino acids. The three-dimensional (3D) model of P. digitatum CYP51 was established based on structure template of 1e9x.pdb and diniconazole was docked into the active site by FlexX. According to spectral data, it is suggested that the purified soluble protein had high affinity with diniconazole, a potent inhibitor of CYP51 reaction in fungi. At the same time, these spectral data suggested that the 3D model and the docking model were reasonable, which we hope can be used to provide a virtual screening of novel DMI drugs.     

Pre-harvest Fungal Colonization Affects Storage Life of Bing Cherry Fruit

Treatment of sweet cherry ( Prunus avium cv. Bing) trees with either two mildewcide cover sprays or multiple (18) applications of topical and systemic fungicides produced cherry fruit that were either highly colonized or relatively uncolonized by fungi, respectively. Fruit from the multiple application treatment had a storage life of up to 8 weeks (7 weeks at 1-4°C plus 1 week at simulated retail temperatures of 20–21°C), whereas the commerciallymanaged fruits (two mildewcides) were extensively colonized by fungi and extensively decayed when stored under the same conditions. Stringent post-harvest disinfection did not significantly reduce postharvest decay, and only slightly reduced isolation frequency of fungi. The data obtained in this study suggest that the degree of preharvest fungal colonization is a primary determinant of the percentage of post-harvest decay, and that pre-harvest colonization can potentially be addressed by pre and post-harvest management practices.     

Reducing the impact of pesticides on biological control in Australian vineyards: pesticide mortality and fecundity effects on an indicator species, the predatory mite Euseius victoriensis (Acari: Phytoseiidae)

Laboratory bioassays on detached soybean, Glycine max (L.) Merr., leaves were used to test 23 fungicides, five insecticides, two acaricides, one herbicide, and two adjuvants on a key Australian predatory mite species Euseius victoriensis (Womersley) in "worst-case scenario" direct overspray assays. Zero- to 48-h-old juveniles, their initial food, and water supply were sprayed to runoff with a Potter tower; spinosad and wettable sulfur residues also were tested. Tests were standardized to deliver a pesticide dose comparable with commercial application of highest label rates at 1,000 liter/ha. Cumulative mortality was assessed 48 h, 4 d, and 7 d after spraying. Fecundity was assessed for 7 d from start of oviposition. No significant mortality or fecundity effects were detected for the following compounds at single-use application at 1,000 liter/ha: azoxystrobin, Bacillus thuringiensis (Bt) subsp. kurstaki, captan, chlorothalonil, copper hydroxide, fenarimol, glyphosate, hexaconazole, indoxacarb, metalaxyl/copper hydroxide, myclobutanil, nonyl phenol ethylene oxide, phosphorous acid, potassium bicarbonate, pyraclostrobin, quinoxyfen, spiroxamine, synthetic latex, tebufenozide, triadimenol, and trifloxystrobin. Iprodione and penconazole had some detrimental effect on fecundity. Canola oil as acaricide (2 liter/100 liter) and wettable sulfur (200 g/100 liter) had some detrimental effect on survival and fecundity and cyprodinil/fludioxonil on survivor. The following compounds were highly toxic (high 48-h mortality): benomyl, carbendazim, emamectin benzoate, mancozeb, spinosad (direct overspray and residue), wettable sulfur (> or = 400 g/100 liter), and pyrimethanil; pyrimethanil had no significant effect on fecundity of surviving females. Indoxacarb safety to E. victoriensis contrasts with its toxicity to key parasitoids and chrysopid predators. Potential impact of findings is discussed.     

Effects of fungicide residues on the survival, fecundity, and predation of the mites Tetranychus urticae (Acari: Tetranychidae) and Galendromus occidentalis (Acari: Phytoseiidae)

Representative fungicides from three or four families used for management of powdery mildew and other diseases in tree fruits were evaluated for their effects on a common spider mite and predator mite species, respectively. A modified Munger cell technique was effective in measuring the response of phytophagous and predaceous mites to fungicide residues on detached leaves in the laboratory. Demethylation-inhibiting (DMI) (imidazole [triflumazole] and triazole [myclobutanil]) and strobilurin (trifloxystrobin) fungicides were not toxic to female Tetranychus urticae Koch and Galendromus occidentalis (Nesbitt), and no sublethal effects were found on fecundity and predation rate after 3-5-d exposure to residues. Benomyl, a benzimidazole fungicide, increased adult mortality and reduced fecundity for both mite species; however, it did not alter the predation rate of G. occidentalis females on T. urticae eggs and larvae. Female G. occidentalis that survived the lethal effects of benomyl and the comparison acaricide pyridaben were unimpaired in predation. Our results for benomyl substantiate those of earlier studies and provide evidence for nontoxic effects of DMI and strobilurin fungicides on mites. We propose that DMI and strobilurin fungicides are a good fit for integrated mite management programs due to conservation of phytoseiid predatory mites.     

Yeast-extract improved biosynthesis of lignans and neolignans in cell suspension cultures of <Emphasis Type="Italic">Linum usitatissimum</Emphasis> L.

Lignans and neolignans are important biologically active ingredients (BAIs) biosynthesized by Linum usitatissimum. These BAIs have multi-dimensional effects against cancer, diabetes and cardio vascular diseases. In this study, yeast extract (YE) was employed as an elicitor to evaluate its effects on dynamics of biomass, BAIs and antioxidant activities in L. usitatissimum cell cultures. During preliminary experiments, flax cultures were grown on different concentrations of YE (0–1000 mg/L), and 200 mg/L YE was found to be optimum to enhance several biochemical parameters in these cell cultures. A two-fold increase in fresh (FW) and dry weight (DW) over the control was observed in cultures grown on MS medium supplemented with 200 mg/L YE. Similarly, total phenolic (TPC; 16 mg/g DW) and flavonoids content (TFC; 5.1 mg/g DW) were also positively affected by YE (200 mg/L). Stimulatory effects of YE on biosynthesis of lignans and neolignans was also noted. Thus, 200 mg/L of YE enhanced biosynthesis of secoisolariciresinol diglucoside (SDG; 3.36-fold or 10.1 mg/g DW), lariciresinol diglucoside (LDG; 1.3-fold or 11.0 mg/g DW) and dehydrodiconiferyl alcohol glucoside (DCG; 4.26-fold or 21.3 mg/g DW) in L. usitatissimum cell cultures with respect to controls. This elicitation strategy could be scaled up for production of commercially feasible levels of these precious metabolites by cell cultures of Linum.     

Influence of pesticides on yeasts colonizing leaves

The effect of nine different pesticides on the growth of yeasts isolated from the leaves of fruit and forest trees was investigated. Four insecticides (with the active ingredients: thiacloprid, deltamethrin, lambdacyhalothrin, and thiamethoxam) and five fungicides (with the effective substances: bitertanol, kresoxim-methyl, mancozeb, trifloxystrobin, and cupric oxychloride) were tested. The concentrations of chemicals were those recommended by the manufacturers for the spraying of trees. The yeast strains isolated from the leaves of fruit trees were not sensitive to any of the insecticides. The majority of yeast strains isolated from the leaves of forest trees were either not sensitive or only to a small extent. While Rhodotorula mucilaginosa and Pichia anomala were not affected by any insecticide, the strains of Cryptococcus laurentii and Rhodotorula glutinis showed the highest sensitivity. The effects of fungicides on the growth of isolated yeasts were more substantial. The fungicide Dithane DG (mancozeb) completely inhibited the growth of all yeasts. All strains isolated from fruit tree leaves were more resistant to the tested fungicides than those isolated from the leaves of forest trees. The most resistant strains from the leaves of fruit trees belonged to the species Metschnikowia pulcherrima, Pichia anomala, and Saccharomyces cerevisiae, whereas Cryptococcus albidus and C. laurentii, originating from the leaves of forest trees, showed the highest sensitivity to fungicides.     

Non-conventional methods for the control of post-harvest pear diseases

Pears are highly perishable products, especially during the post-harvest phase, when considerable losses can occur. Among the fungal diseases, blue mold caused by Penicillium expansum, grey mould caused by Botrytis cinerea, Mucor rot caused by Mucor piriformis are common on pear fruits. Other (weak) pathogens like Phialophora malorum, Alternaria spp., and Cladosporium herbarum tend to infect wounds and senescent fruits. A post-harvest fungicide treatment can reduce decay but effectiveness decreases with the appearance of resistant strains. There is a clear need to develop new and alternative methods of controlling post-harvest diseases. The emerging technologies for the control of post-harvest fungal diseases are essentially threefold: application of antagonistic microorganisms, application of natural antimicrobial substances and application of sanitizing products. Two biological control products, Aspire (Candida oleophila I-182) (Ecogen, Langhorne, PA, USA) and Bio-Save 110 (Pseudomonas syringae) (EcoScience, Worcester, MA, USA; formerly Bio-Save 11) are currently registered in the USA for post-harvest application to pears. Other potential biocontrol agents have been isolated from fruit and shown to suppress post-harvest decay in pear. It is important that evaluation of these microorganisms be carried out in a product formulation because the formulation may improve or diminish antagonistic efficacy depending on the concentration of chemical product and the duration of exposure to the treatment. Plants produce a large number of secondary metabolites with antimicrobial effects on post-harvest pathogens. Detailed studies have been conducted on aromatic compounds, essential oils, volatile substances and isothiocyanates, with encouraging results. In particular, allyl-isothiocyanate used as a volatile substance, controls blue mould in 'Conference' and 'Kaiser' pear inoculated with a thiabendazole-resistant strain. Sanitizing products such as chlorine dioxide, peracetic acid and ozone have considerable fungicidal activity against P. expansum and M. piriformis, depending on the concentration of chemical product and the duration of exposure to the treatment. Sanitizing solutions can be integrated easily with current handling and storage practices; however, further research is required to define the effective procedures better.     

Influence of curing times on the effectiveness of treatments with acetic acid on the control of P. digitatum on lemons

The restricted number of postharvest fungicides used in packing houses is leading to the selection of resistant strains of Penicillium digitatum (citrus green mould), one of the most common and serious pathogens during storage and marketing of lemons. Furthermore a growing concern for human health and a greater awareness for environmental conservation have multiplied the studies on new ecological technologies. Among the alternatives to synthetic postharvest fungicides, the use of acetic acid (classified as GRAS) together with a physical method such as curing, have led to encouraging results. In the present study is reported the combined use of curing, performed at reduced times compared to those reported to be effective, followed by acetic acid (AAC) treatments. Lemons of the variety "Limone di Massa" artificially inoculated with P. digitatum at a concentration of 10(4) spores/mL were cured for 0, 3, 6, 12 and 24 hours and then treated with three different concentrations of AAC (25, 50 and 75 microL/L) for 15 min. Fruit was then stored at 20 degrees C and 80% relative humidity (RH) for 9 days, when the number of decayed fruits was monitored. The same combined treatments were also carried out on naturally infected lemons, stored for 6 or 8 weeks at 5 degrees C and 90% RH. After 9 days of storage the lowest percentage of infected wounds, in artificially inoculated fruit, was 0% after 6 hours of curing followed by AAC fumigation performed at 50 microL/L, while lemons untreated or cured for three hours showed the worst results with 71.4 and 61.9% of rotted fruit respectively. In naturally infected lemons the best results were achieved with curing performed for 24 hours followed by AAC fumigation at 50 microL/L. In these cases the combined treatment reduced decay by the 91.0 and 66.5% after 6 or 8 weeks of storage respectively, if compared to untreated fruit. The weight loss was not affected by any of the treatments. These results show that a good control of green mould during storage could be achieved, on lemon fruit, by combining a reduced curing time of 24 hours to the effect of AAC. The best results were obtained after 6 week of storage even if a satisfactory control was observed after 8 weeks of storage.     

桃ACC合酶基因的分离及其差异表达

利用5′/3′RACE PCR技术,从桃(Prunus persica (L.) Batsch)果实中克隆了植物乙烯生物合成的关键酶--ACC合酶的全长cDNA pacs,对pacs基因进行全序列测定表明,该基因全长1 848个碱基,编码区为1 449个碱基,5′端有177个碱基的非编码区序列,3′端有219个碱基的非编码区序列(不包括终止密码子TAA).pacs基因编码区共编码483个氨基酸,蛋白质大小为54 kD,等电点为6.43.pacs与番茄(S19677)、梅(AB031026)、番木瓜(U68216)、苹果(AB034993)等其他植物ACC合酶cDNA氨基酸序列同源性分别为65%、70%、75%、90%,并存在与这些ACC合酶氨基酸的活性位点保守序列SLSKDMGFPGFR.RT-PCR结合杂交分析表明,pacs和我们以前克隆的桃ACC合酶cDNA pacs12(AF467782)在叶片和花中基因表达模式基本一致,伤处理和IAA均能诱导叶片pacs 和pacs12基因的表达,但pacs在伤处理叶片的表达水平比pacs12高;pacs 和pacs12基因在果实表达有所不同,pacs在绿熟和成熟果实中均有表达,而pacs12在绿熟果实中基本检测不到,在成熟果实中才有表达,两者在果实中的表达水平比伤处理和IAA处理叶片和花中要低.