A biomarker for the identification of four Phaeoacremonium species using the β-tubulin gene as the target sequence

A real-time polymerase chain reaction (PCR) was developed for the rapid detection and identification of Phaeoacremonium species, the fungi associated with severe diseases in grapevines. A degenerate primer pair (F2bt-R1bt) with homology to the β-tubulin gene was designed to be used in the amplification of 11 species of Phaeoacremonium. Four species-specific probes labelled with three different fluorescent dyes were designed to be used with the degenerate primers in a real-time PCR for the identification of Phaeoacremonium aleophilum, P. parasiticum, P. viticola and P. mortoniae. Combinations of two probes in a duplex real-time PCR allowed to detect and identify a mixture of Phaeoacremonium species and cross-amplifications were not detected. This method was applied to detect Phaeoacremonium species in eight wood fragments from grapevine plants naturally infected, and results were compared with those obtained with nested PCR and culturing on growth media. Real-time PCR detected Phaeoacremonium in 100% of the analysed fragments, whereas nested PCR did only in the 62% of them and requiring subsequent restriction fragment-length polymorphism analysis to identify the species. This method is a sensitive tool to detect and identify Phaeoacremonium species in infected grapevine wood. Real-time PCR assay defined here can be used in a plant nursery program to identify pathogen-free plants in order to manage Petri disease of grapevines. © 2008 Springer-Verlag

Genetic diversity in Botrytis cinerea populations on vegetable crops in greenhouses in south-eastern Spain

Seventy-nine random amplified polymorphic DNA (RAPD) markers, of which 46 were polymorphic and 33 monomorphic, were used to determine the level of generic diversity and to study the structure of populations of Botrytis cinerea in Almeria, Spain. Sixty-five isolates were collected in the first survey (December 1992, at the beginning of the epidemic) from 23 commercial greenhouses of common winter crops in two regions, Levante and Pomente. In the second survey, conducted three months later at the end of the epidemic, 75 isolates were collected from 16 greenhouses in Poniente. The analysis of population structure revealed that generic diversity within subpopulations sampled in the two surveys accounted for 98% of the total generic diversity, while generic diversity between subpopulations represented only 2% of the total diversity. The relative magnitude of gene differentiation between subpopulations, G(ST), averaged 0-017. In the first survey, partition of generic diversity into hierarchical components by greenhouses within regions showed that the within-greenhouse component of diversity was 93%, the between-greenhouses, within-region component was 6%, and the between-regions component was 1%. Similar results were obtained when comparisons were made between greenhouses in the second survey or between populations of different countries genetic diversity within subpopulations accounted for 95 and 96%, respectively, of the total genetic diversity. In all comparisons, values of G(ST) were higher than 0.01, ranging from 0.011 to 0.055. Analysis of likelihood ratio chi-square (G2) statistics under the null hypothesis that frequencies of markers are similar between subpopulations showed that different markers contributed to the differentiation between subpopulations in each comparison. The relative importance of several evolutionary forces in populations of B. cinerea is discussed, together with implications for the management of grey mould

Survival of Botrytis cinerea in southeastern Spanish greenhouses

The relative importance of sclerotia and mycelia of Botrytis cinerea Pers ex Fr. as structures of survival in southeastern Spanish greenhouses was investigated. Sclerotia were not found in the SE region, neither on plant debris nor on living plant material, suggesting it may serve only a minor role in epidemic development. B. cinerea survived mostly as mycelium. The percentage of artificially inoculated tomato stem pieces from which mycelium was recovered, was used to quantify its survival rate. Outside the greenhouses, mycelium survived in 33% and 5% of the tomato stem pieces 110 days after inoculation in 1995 and 1997, respectively. After the same number of days inside the greenhouses, no mycelium was recovered from stem pieces in 1995, and in 1997 only 7% of the stem pieces contained mycelium. Survival of mycelium outside and inside the greenhouses was significantly (P < 0.05) different after 47, 83, and 110 days of exposure to field conditions in 1995, but they were not different in 1997. Under controlled conditions, mycelium of B. cinerea lost viability at 100% relative humidity at temperatures ranging from 5 to 40°C, suggesting that air temperature and relative humidity accounted for loss of viability of mycelium

Fitness of Botrytis cinerea associated with dicarboximide resistance

Fitness costs in Botrytis cinerea associated with dicarboximide resistance were studied. Spearman rank correlation coefficients were calculated between resistance to iprodione and survival ability both outside and inside the greenhouse, measured on isolates randomly chosen from a collection done in a survey of commercial greenhouses in Southeastern Spain in 1992. Survival was measured at 47, 83, and 110 days as percentage of surviving mycelia in a sample of artificially inoculated tomato stem pieces and as percentage of viable sclerotia from a sample of sclerotia produced on potato dextrose agar. Resistance to iprodione was measured by the fungicide concentration that reduces fungal growth by 50% (EC50 values). Significant (P &lt; 0.05) negative correlation coefficients between survival of sclerotia and resistance to iprodione were found for some samplings dates, which indicates that sclerotia of resistant isolates survive less well than sclerotia from sensitive isolates. For mycelia, no relationship between survival and resistance was found

Integrated Botrytis cinerea management in southeastern Spanish greenhouses

A strategy for integrated biological and chemical control of Botrytis cinerea in unheated tomato greenhouses in southeastern Spain was tested. The biocontrol agent used was a commercial preparation developed from an isolate of Trichoderma harzianum T39 (Trichodex®). Decisions concerning whether to spray the biocontrol agent or a fungicide were made based on a future weather disease warning system called BOTMAN implemented as follows no spraying when slow or no disease progress was expected; use of a chemical fungicide when an outbreak of epidemics was expected; in all other cases, application of Trichoderma harzianum T39 was recommended. A 4-day weather forecast provided by the Eastern Andalusian Weather Forecast Service was used for predictions. The integrated strategy was compared with weekly applications of fungicides in two experiments conducted over 1998-99 and 1999-2000. Reduction of disease incidence was obtained only with the fungicide-only treatment in the 1998-99 experiment (55%, P<0.05). Application of BOTMAN gave high disease risk only in 2 dates or times in that experiment, so fungicides were only applied twice. For the remaining 12 dates or times, Trichodex® was sprayed. In the second experiment, application of BOTMAN gave moderate risk all the weeks and Trichodex® was applied nine times. In this experiment, disease level did not differ significantly (P < 0.05) from untreated plot. The reasons for failure of BOTMAN in Spanish conditions were discussed

Storage of Botrytis cinerea using different methods

Different methods were used to store a collection of field isolates of B. cinerea. Five isolates each of the fungus were stored (i) in silica gel at 4°C, (ii) in sand at 4°C, (iii) in potato dextrose agar slants at 4°C, (iv) as dry spores in tubes at -20°C, or (v) as spores in glycerol (20%) at -20°C. Different characteristics were tested after 1, 2 and 4 years of storage. Viability was poor when spores were stored dried, and contamination was frequent when stored in potato dextrose agar. Linear growth rates of cultures stored for 4 years were similar to initial values, except for one isolate in sand (9% reduction) and for another in glycerol (14% reduction). Sclerotia size and sporulation were reduced after 4 years of storage, independently of the method used. Fungicide resistance to procymidone changed to sensitive in all the isolates, except for one isolate, after four years of storage, also regardless of the storage method used. Storage in sand at 4°C or in glycerol at -20°C were the best methods for preserving B. cinerea isolates

Distribution and fitness of isolates of Botrytis cinerea with multiple fungicide resistance in Spanish greenhouses

Forty-nine greenhouses of vegetable crops were surveyed in southeast Spain at the beginning of the disease season in December 1992 to estimate frequencies of resistance to benzimidazoles, dicarboximides and N-phenylcarbamates (NPC) in B. cinerea. Out of 261 isolates collected, 28% were sensitive to both benzimidazoles and dicarboximides, 15% were benzimidazole-resistant and dicarboximide-sensitive, 8% were benzimidazole-sensitive and dicarboximide-resistant and 46% were benzimidazole- and dicarboximide-resistant. Resistance to benzimidazole, dicarboximide and N-phenylcarbamate was determined by measuring the ability of each isolate to grow in the presence of carbendazim, procymidone and diethofencarb fungicides respectively. Carbendazim- or procymidone-resistant isolates were found in all surveyed greenhouses. Three isolates were found with resistance to carbendazim, procymidone and diethofencarb collected in two adjacent greenhouses that were sprayed with the carbendazim and diethofencarb mixture. All other isolates were sensitive to the mixture because they were either sensitive to carbendazim and resistant to diethofencarb or vice versa. Fitness of 31 isolates of B. cinerea was determined in vivo by measuring their sporulation and lesion growth rate on leaf disks. No fitness costs were associated with resistance to iprodione (dicarboximide) and benomyl (benzimidazole). Isolates with EC50 values higher than 101 mg/L for benomyl and 1.6 mg/L for iprodione were considered to be field resistant (they caused visible lesions on cucumber leaf disks treated with each fungicide)

Comparison of RAPD and AFLP marker analysis as a means to study the genetic structure of Botrytis cinerea populations

To assess the genetic relationships of Botrytis cinerea populations in Almería (Spain), 44 isolates of B. cinerea, collected from six commercial greenhouses (subpopulations), were analysed by Random Amplified Polymorphic DNA (RAPD) and amplified-fragment length polymorphism (AFLP). Polymorphisms were more frequently detected per primer with AFLP than with RAPD (16 compared to 4). However, RAPD detected polymorphisms more frequently per loci than AFLP (56% compared to 32%). The analysis of population structure revealed that the genetic diversity within subpopulations (HS) accounted for 96% of the total genetic diversity (HT), while genetic diversity among subpopulations represented only 4% of the total diversity, independently of whether they were analysed with RAPD or AFLP markers. The relative magnitude of gene differentiation between subpopulations (GST) and the estimate of the number of migrants per generation (Nm) averaged similar values when estimated with RAPD or AFLP markers (0.039 and 0.036, or 12.32 and 13.39, respectively). The results obtained in dendrograms were in accordance with the gene diversity analysis. However, the diversity of B. cinerea was higher when analysed by RAPD than with AFLP. In these cases, the isolates could not be grouped by greenhouse or fungicide resistance (except those sensitive to carbendazim and resistant to procymidone). Both the RAPD and AFLP technologies are suitable for studies of genetic structure or B. cinerea populations, although RAPD generated more polymorphisms per loci than AFLP, and provided a better explanation of the genetic relationships between isolates