Evidence for a constitutive cytoplasmic cutinase in ungerminated conidia of Botrytis cinerea Pers.: Fr.

Cytoplasmic soluble proteins from ungerminated conidia of Botrytis cinerea exhibited cutinase activity, while cell wall binding proteins lacked this activity. Cutinase activity in proteins extracted from cell walls and cytoplasm of ungerminated conidia of Botrytis cinerea was determined using p-nitrophenyl butyrate (PNB) and TLC analysis of products derived from hydrolysis of [³H]cutin. Treatment of conidia with indoxyl acetate, a substrate indicative of non-specific esterase and cutinase activity, also gave a positive reaction in the cytoplasm of ungerminated conidia. The possible role of a putative constitutive cutinase in the cytoplasm of conidia in the early stages of infection of plants by B. cinerea is discussed.     

Effects of long-term storage at different temperatures on conidia of Botrytis cinerea Pers.: Fr.

Survival of Botrytis cinerea conidia was studied after storage without pretreatments at different temperatures (-80 degrees C, -20 degrees C, 4 degrees C and 21 degrees C). Germination tests performed during 3 years showed that viability at 21 degrees C was completely lost after 1 month. Conidia stored for 30 months at -80 degrees C, -20 degrees C and 4 degrees C were able to germinate, respectively, at 79%, 8% and 0.2%. Changes in adenylate level, energy charge and respiration (O(2) consumption) made on each set of conidia were correlated to the germination rate. The 30-month-old stored conidia showed differences in pathogenicity tests on apples. While the pathogenic aggressiveness of conidia stored at -80 degrees C was almost the same as for fresh conidia, it decreased with increasing temperature of storage. An ultrastructural study made on conidia stored for 30 months at -80 degrees C has shown the emergence of a new wall layer in a retraction zone of the cytoplasm by comparison to fresh conidia. However, the integrity of the cytoplasmic content was maintained. The effects of low temperature storage, maintenance of cell integrity and pathogenicity of conidia of B. cinerea are discussed.     

Purification and characterization of a 40.8-kDa cutinase in ungerminated conidia of Botrytis cinerea Pers.: Fr

Cytoplasmic soluble proteins from ungerminated conidia of Botrytis cinerea exhibited cutinase activity. A 40.8-kDa cutinase was purified to homogeneity from this crude conidial protein extract. This cutinase does not correspond either to constitutive or to induced lytic cutin enzymes already described by other authors. The possible role of this constitutive cutinase in the induction of other cutinolytic proteins in the early stages of infection of plants by B. cinerea is discussed.     

Characterization of FaRB7, a near root-specific gene from strawberry (Fragariaxananassa Duch.) and promoter activity analysis in homologous and heterologous hosts

Many insect and fungal pathogens posing agronomically important threats specifically target the roots in strawberry. The use of a root-specific promoter to confer expression of resistance genes in a targeted manner has the potential appreciably to benefit the genetic improvement of commercial strawberry varieties. A novel gene, FaRB7, was isolated from strawberry (Fragariaxananassa Duch.) and found to contain motifs characteristic of tonoplast intrinsic proteins (TIPs). Phylogenetic analysis revealed that FaRB7 represents an RB7-type TIP. In strawberry, this gene is expressed predominantly in roots, with very low expression in petioles. A 2.843 kb region representing the FaRB7 gene upstream regulatory sequence was isolated and found to share a number of sequence motifs with the promoter of the Nicotiana tabacum TobRB7 root-specific RB7-type TIP. When cloned upstream of the gusA reporter gene and introduced into strawberry plants, the FaRB7 promoter was shown to direct strong, near root-specific expression with expression patterns very similar to that of the endogenous gene. Furthermore, the FaRB7 promoter was found to confer constitutive expression, comparable to that produced by the cauliflower mosaic virus (CaMV) 35S RNA promoter, in tobacco. Thus, the FaRB7 promoter may be used to achieve near-root-specific transgene expression in strawberry and also represents an alternative to the CaMV 35S promoter for producing constitutive foreign gene expression in heterologous hosts. The FaRB7 full-length genomic sequence and 5' upstream regulatory region have been submitted to the EMBL/GenBank database under accession number DQ178022.     

Biological Control of Botrytis cinerea Pers. ex Fr. in Strawberry by Paenibacillus polymyxa (Isolate 18191)

Isolate 18191, obtained from mature strawberry fruit and determined as Paenibacillus polymyxa has shown an antagonistic potential against Botrytis cinerea , the causal agent of grey mould in strawberries. Germ tube growth of conidia of B. cinerea was strongly inhibited by the culture suspension of the antagonist in aqueous strawberry fruit pulp suspension (1%) but germination rate of conidia was not affected. The application of the culture suspension and the washed cells on detached strawberry leaf discs reduced conidiophore density of B. cinerea by 67 and 84%, respectively. The treatment of detached leaf discs with culture suspensions of different cell densities (1 × 10⁶, 1 × 10⁷, 1 × 10⁸) showed that the lowest density already reduced incidence of B. cinerea by 68% after 8 days incubation period. Investigating the influence of the temperature on the effectiveness of P. polymyxa it was observed that the antagonist was highly effective already at 10°C and reduced incidence and conidiophore density of B. cinerea by 53 and 58%, respectively. In 3-year field trials the effectiveness of P. polymyxa was in a range of 24–36% as compared to the water control.     

Involvement of ethylene in the regulation of growth and development of the fungus Botrytis cinerea Pers. ex. Fr.

Application of ethephon slightly increased the growth of hyphae of Botrytis cinerea. A competitive inhibitor of ethylene binding, 2,5-norbornadiene (NBD), inhibited growth of hyphae and mycelium and retarded the development of Botrytis cinerea. Transfer of the mycelium from an atmosphere containing NBD to air relieved the inhibition, indicating that the NBD effects were non-toxic and reversible. Addition of exogenous ethylene to an atmosphere containing NBD (20 ml 1^-1) effectively reduced the inhibition. Inhibition due to 40 ml 1^–1 NBD was not relieved by ethylene at any of the concentrations tested; however, a positive effect of ethylene appeared following transfer of the mycelia to air. The results suggest that ethylene may be required for the growth and development of Botrytis cinerea.Abbreviations NBD 2,5-norbornadiene - ethephon 2-chloroethyl-phosphonic acid - PDA potato dextrose agar     

Local and systemic protection of poinsettia (Euphorbia pulcherrima Willd.) against Botrytis cinerea Pers. induced by benzothiadiazole

Benzothiadiazole (BTH) was found to be highly effective in increasing resistance of two poinsettia cultivars — ‘Coco White’ and ‘Malibu Red’, moderately susceptible to the fungus Botrytis cinerea. BTH applied at a concentration of 0.3 mM on the discs cut out from the leaves of these poinsettia cultivars reduced disease symptoms by more than 60 % in comparison to the control discs treated with water and exposed to infection. It was also observed that the applied inducer at a concentration of 0.03 and 0.3 mM had a favourable influence on the increase of poinsettia systemic resistance of SAR type (systemic acquired resistance). The effectiveness of BTH was much less when disease development was examined on detached leaves (a 20 % reduction of lesion area) in comparison with a pronounced inhibition of grey mould development on intact leaves of previously induced plants (a 80 % protection of intact plants). Benzothiadiazole in the concentration range from 0.03 to 1.4 mM added to in vitro agar medium was not found to have an inhibitory influence on Botrytis cinerea mycelium growth and sporulation.     

Development of real-time PCR (TaqMan) assays for the detection and quantification of Botrytis cinerea in planta.

Real-time PCR assays based on TaqMan chemistry have been developed for the detection and quantification of Botrytis cinerea, suitable for a wide range of different host plant species. Assays were designed to the beta-tubulin gene, the intergenic spacer (IGS) region of the nuclear ribosomal DNA and also to a previously published, species-specific sequence characterised amplified region (SCAR) marker; the assays were compared to a published method based on SYBR Green I technology. The assays designed to the IGS region and SCAR marker proved to be highly specific for B. cinerea but assays designed to the beta-tubulin gene and the previously published assay designed to the cutinase-A gene both cross-react with B. fabae. The assay designed to the IGS region was the most sensitive and was able to reliably detect and quantify as little as 20 fg of B. cinerea DNA. The method incorporates the detection of a gene from the plant host to compensate for variations in extraction efficiency and size of sample tested. The assays designed were used to follow the progression of infection of B. cinerea in plant material inoculated with spores to the point of symptom induction. They should be ideally suited to investigating infection processes in-planta and could be used to investigate aspects of infection/plant pathogenesis, by B. cinerea and are particularly suited to the detection and quantification of the pathogen prior to the development of symptoms.     

Mycobiota and molecular detection of Aspergillus flavus and A. parasiticus aflatoxin contamination of Strawberry (Fragaria ananassa Duch.) fruits

Strawberry fungi were isolated from fresh fruits and juice on the two types of media (Sabouraud dextrose agar, SDA and potato-dextrose agar, PDA) at 28 °C. Nineteen fungal species belong to 12 genera were isolated from fruits and juice on both isolation media. The most common fungal genera and species were Aspergillus flavus, A. niger, Mucor racemosus, Neurospora crassa, Penicillium chrysogenum, Rhizopus stolonifer and Trichoderma harzianum. Twenty A. flavus and A. parasitics isolates were assayed for their abilities to produce aflatoxins. The concentration of aflatoxins ranged between 25.8–75.2 and 23.6–71.1 ng/ml at 350 and 365 nm, respectively. Among A. flavus and A. parasiticus strains tested, aflatoxin B contributed 30–60% of total isolates. However, G type contributed 85–90%. The R_f values of B₁, B₂, G₁ and G₂ were 0.79, 0.61, 0.44 and 0.32, respectively. High-performance liquid chromatography analysis of extracts revealed the presence of aflatoxins with variable levels.     

Development of a new probe for specific and sensitive detection of 'Candidatus Phytoplasma mali' in inoculated apple trees

A new TaqMan minor groove binding (MGB) probe and new PCR conditions were designed for quick, specific and sensitive detection of ‘Candidatus Phytoplasma mali’. The new probe can distinguish a single mismatch between ‘Ca. P. mali’ and ‘Candidatus Phytoplasma prunorum’, this constituting an improvement over a previously published method. In our study, the relative position of the mismatch in the MGB probe influenced greatly the specificity of detection. Our new real‐time PCR protocol was able to detect one plasmid copy in water and 100 copies in healthy plant DNA extracts. The sensitivity of this new real‐time PCR method, three other real‐time PCR protocols and a conventional PCR with fU5/rU3 primers was compared. Periwinkles and MM106 rootstocks were grafted with infected material and surveyed over time by symptom observation, conventional PCR and real‐time PCR. Phytoplasma infection was detected by symptom observation in all periwinkles within 4 months and in 75% of the MM106 rootstocks by the end of 7 months. Conventional PCR detected phytoplasma infection in all periwinkles within 4 months and in all MM106 rootstocks within 7 months. Best results were obtained by our real‐time PCR, which detected phytoplasma infection in all grafted plants within 3 months after inoculation.     

Latency of Botrytis cinerea Pers.: Fr. and Biochemical Studies During Growth and Ripening of Two Grape Berry Cultivars, Respectively Susceptible and Resistant to Grey Mould

Latency and development of Botrytis cinerea were assessed under field conditions and after artificial inoculation of two grape varieties, Gamay (susceptible) and Gamaret (resistant). When the percentage of latent Botrytis was the same for both varieties, severity of visible grey mould remained very low in Gamaret berries, while Gamay clusters were destroyed by the disease to a high percentage. Some biochemical parameters were measured in berries, such as constitutive and induced anti‐fungal compounds, polymeric proanthocyanidins and lipid peroxidation products as markers of senescence. Differences were observed in polymeric proanthocyanidins (PPRA) of Gamaret compared with those of Gamay. Concentration and mean degree of polymerization (mDP) of PPRA were always higher in the berries of the resistant variety. The inhibitory effect of Gamaret PPRA on enzyme activity remained until harvest whereas Gamay PPRA lost their inhibitory activity at the beginning of véraison. Based on these results, resistance to B. cinerea seems to be linked to the maintainance of the fungus in its latent form in berries, mainly due to the ability of Gamaret PPRA to inhibit macerating fungal enzyme activities.     

基于量子点标记的赭曲霉毒素A快速、高灵敏荧光免疫层析检测方法的建立及应用

赭曲霉毒素A（ochratoxin A,OTA）具有肾毒性、致畸性、致癌性和免疫毒性,广泛存在于各种粮食作物及其副产品中,是食品和饲料原料的重要污染物,可在人类及动物体内蓄积,在已知发现的真菌毒素中,重要性和危害性仅次于黄曲霉毒素。本研究通过采用量子点荧光微球（quantum dots,QDs）标记OTA单克隆抗体,并基于免疫层析原理,优化、建立了OTA高灵敏荧光免疫层析检测方法（FICGA）,15min即可实现对农产品中OTA污染的快速定量检测。该方法检测下限（IC₁₀）达到0.04ng/mL,检测区间（IC₂₀-IC₈₀）为0.05-0.59ng/mL,半数抑制率（IC₅₀）为0.18ng/mL。与OTA类似物OTB、OTC交叉反应性为7.3%和11.9%,对其他常见真菌毒素AFB₁、ZEN、FB₁和DON均无交叉反应。在玉米、面粉和大豆样本中的加标回收率可达83.2%-117.8%,与LC-MS/MS同时对天然样本中OTA含量的检测结果表明,两种方法相关性良好。本研究建立的FICGA快速、灵敏,可满足基层单位和现场的快速检测需求,具有很好的应用前景。     

Sandwich microgravimetric immunoassay: sensitive and specific detection of low molecular weight analytes using piezoelectric quartz crystal

A multi-channel sandwich microgravimetric immunoassay (sMIA), using the quartz crystal microbalance (QCM) principle, has been developed to quantify low molecular weight substances in standard solutions. An antigen is sandwiched between two antigen-specific antibodies: the first antibody is coated on the quartz crystal surface and the second antibody is used for the detection of analyte. The concentration of low molecular weight antigen (insulin was used in this study, M _r6000 Da) was correlated with the shift of resonant frequency of QCM system before and after second antibody binding to insulin. The developed assay is highly specific showing low cross-reactivity, and is sensitive to approx. 1 ng insulin ml^–1 with a linear response for insulin from 10 g ml^–1 to 10 ng ml^–1 in standard solutions. The technique may also be applied for the detection of other small biomolecules.     

食品中沙门氏菌分子检测靶点的筛选与评价

[目的]发掘新的沙门氏菌分子检测靶点,筛选检测性能优秀的引物.[方法]利用BLAST程序比较沙门氏菌属内基因组DNA序列的同源性以及沙门氏菌与非沙门氏菌基因组DNA序列之间的特异性,发掘出100多个检测沙门氏菌属的特异性片段,并从中随机挑选出15个片段作为候选靶点,一共设计了27对引物(FS1～FS27),对它们的特异性、灵敏度加以评价,从中筛选检测性能最好的引物.[结果]在27对引物中,检测性能最优的引物为FS23,采用该引物对供试菌株的相应检测靶点进行PCR扩增,44株沙门氏菌都能扩增到一条492 bp特异性片段,而22株非沙门氏菌则不能扩增出这一特异性片段.以FS23为引物建立PCR方法检测猪霍乱沙门氏菌基因组DNA的灵敏度为11.9 fg/μL,细菌纯培养物灵敏度为4.9×102cfu/mL;用猪霍乱沙门氏菌人工污染牛奶样品,如果接种起始菌量为100 cfu/25 mL时,只需要增菌5 h,采用上述方法即能检测出沙门氏菌.[结论]引物FS23对应的基因序列是一个性能优良的新分子检测靶点,具备很高的特异性和灵敏性,能够广泛应用于食品中沙门氏菌的快速检测.     

Molecular identification of Botrytis cinerea,Botrytis paeoniae and Botrytis pseudocinerea associated with gray mould disease in peonies (Paeonia lactiflora Pall.) in Southern Chile

BackgroundIn Chile, the peony is the most important ornamental flower exported from the country. Gray mould is a phytopathological problem of this crop. This disease is caused by Botrytis cinerea and Botrytis paeoniae.AimsWe carried out the first survey of Botrytis species associated with peony gray mould in Southern Chile to estimate the diversity of these pathogens.MethodsDiseased peony leaves were collected from seven locations in Southern Chile covering a distance of 300 km. The Botrytis isolates obtained were studied by morphological and molecular methods. Finally, a PCR assay using primers based on the necrosis and ethylene-inducing protein gene (nep1) was used to specifically identify B. paeoniae.ResultsSeventeen isolates belonging to Botrytis genus were obtained, and all of them were pathogenic to peonies when inoculated in plants grown in a greenhouse. Morphological analyses showed that four isolates shared common characteristics, which distinguish them from the rest. Homology and phylogenetic analysis of G3PDH, as well as determination of the Bc-hch allele, allowed us to identify 12 isolates as B. cinerea, 4 as B. paeoniae and one isolate as Botrytis pseudocinerea. The PCR assay was found to be specific to B. paeoniae, amplifying a single band of 470 bp.ConclusionsThree Botrytis species involved in peony gray mould disease are present in Chile. This is the first time that both B. paeoniae and B. pseudocinerea have been reported to be present in the country and also that they affect peonies. Finally, to our knowledge, the PCR based method herein described is the first of its kind to be used to identify B. paeoniae.     

Improving PCR detection of prey in molecular diet studies: importance of group‐specific primer set selection and extraction protocol performances

While the morphological identification of prey remains in predators' faeces is the most commonly used method to study trophic interactions, many studies indicate that this method does not detect all consumed prey. Polymerase chain reaction–based methods are increasingly used to detect prey DNA in the predator food bolus and have proven efficient, delivering highly accurate results. When studying complex diet samples, the extraction of total DNA is a critical step, as polymerase chain reaction (PCR) inhibitors may be co‐extracted. Another critical step involves a careful selection of suitable group‐specific primer sets that should only amplify DNA from the targeted prey taxon. In this study, the food boluses of five Rattus rattus and seven Rattus exulans were analysed using both morphological and molecular methods. We tested a panel of 31 PCR primer pairs targeting bird, invertebrate and plant sequences; four of them were selected to be used as group‐specific primer pairs in PCR protocols. The performances of four DNA extraction protocols (QIAamp^® DNA stool mini kit, DNeasy^® mericon food kit and two of cetyltrimethylammonium bromide‐based methods) were compared using four variables: DNA concentration, A₂₆₀/A₂₈₀ absorbance ratio, food compartment analysed (stomach or faecal contents) and total number of prey‐specific PCR amplification per sample. Our results clearly indicate that the A₂₆₀/A₂₈₀ absorbance ratio, which varies between extraction protocols, is positively correlated to the number of PCR amplifications of each prey taxon. We recommend using the DNeasy^® mericon food kit (QIAGEN), which yielded results very similar to those achieved with the morphological approach.     

Mining and evaluation of new specific molecular targets for the PCR detection of Salmonella spp. genome

The purpose of this study is to find new molecular targets for the detection of Salmonella. With the online BLAST Program, we compared homology of genomic sequences and specificity in GenBank among Salmonella serovars and non-Salmonella strains and found 98 Salmonella specific target sequences. We selected 33 target sequences of Gene ID from 3335000 to 3337003 for the specificity evaluation, and finally 8 specific fragments screened out, they are 3334138, 3335583, 3335471, 3335211, 3335068, 3336466, 3336736 and 3336998. Primer SC8 of gene 3335583 and SC9 of gene 3335471 were the best in specificity and sensitivity among these primers. The detection sensitivity of Primer SC9 was 1.23 fg/μl for DNA templates and 720 cfu/ml for whole cells, while primer SC8’s was 12.3 fg/μl and 720 cfu/ml, respectively. Salmonella could be detected successfully by the PCR method developed in this study after 8 h enrichment when the milk samples were artificially contaminated by this organism at 7 cfu per 10 ml milk.