Observation on Baseline Sensitivity of Erysiphe necator Genetic Groups to Azoxystrobin

Powdery mildew, caused by Erysiph necator, is a common and severe fungal disease of grapevine all over the world. The disease costs millions of dollars to vine growers, due to intensive use of fungicides and yield losses. Recently in population of E. necator two genetic groups have been described, the two groups seem to occupy different temporal niches, with a temporal alternation that is clear-cut in vineyards intensively treated with chemical fungicides. QoISTAR (Quinol Outside Inhibitors-Strobilurin Type of Action and Resistance) fungicides are widely used to control the disease, and generally carry a high risk of pathogen resistance development. To clarify the behaviors of the biotrophic fungus when treated with azoxystrobin as a representative of QoI-STAR, baseline sensitivity of laboratory isolates were determined. A leaf bioassay and the primers RSCBF1 and RSCBR2 designed on the highly conserved regions of cytb gene in fungi were used. Partial sequence of E. necator cytb gene were obtained. Attempts to obtain a laboratory mutant were not totally successful. The sensitivity to azoxystrobin (EC50) in isolates of genetic group B was significantly higher than in isolates of group A, to which all the isolates collected later in the season belonged. The higher sensitivity to azoxystrobin fungicides observed in group B isolates can be at the basis of their precocious disappearance in vineyards, and can have important implications for powdery mildew control strategies.


Introduction
Powdery mildew, caused by the obligate fungus Erysiphe necator Schw., is a common and severe fungal disease of grapevine worldwide, due to the high adaptability of the pathogen to different climatic conditions.In populations of the fungus, two genetic groups or biotypes corresponding to the different overwintering forms have been described (quiescent mycelium in buds, "flag shoot" biotype (group A) and cleistothecia, "ascospore" biotype (group B) [1].Alternation of the two groups is clear-cut evidence in vineyards intensively treated with chemical fungicides, where group B isolates can persist throughout the season [1].Thus, changes in the composition of fungal populations might be influenced by spray schedules as a result of different sensitivity to fungicides [2].
Control of the disease is exclusively depending on intensive application of fungicides.Quinol Outside Inhibitors-Strobilurin Type of Action and Resistance (QoI-STAR) fungicides are one of the most important class of agricultural fungicides that are widely used to control grapevine powdery mildew.These fungicides inhibit mitochondrial respiration at the ubiquinol oxidation cen-tre (Qo site) of the cytochrome bc1 enzyme complex (complex III) [3].Because of their single-site mode of action, QoI fungicides generally carry a high risk of pathogen resistance in more than 30 phytopathogenic species, such as casual agent of powdery mildews, downy mildews, anthracnose, scab, and grey mould [4].
Two mechanisms of resistance to QoI fungicides are known.The first involves one or several point mutations in the cytb gene, resulting in changes of the peptide sequence preventing fungicide binding [5].Single or combined point mutations in the cytochrome b gene (cytb) were detected in many fungi at amino acid position 127 to 147 and 275 to 296 [6,7].Substitution of glycine with alanine at position 143 (G143A) was described to be associated with the expression of high resistance, while substitution of glycine to arginine at position 137 (G137R) and substitution of phenylalanine to leucine at position 129 (F129L) are associated with the low resistance [4,8].The second mechanism was observed in vitro and involves's the activity of alternative oxidase (AOX) enzyme, which oxidise ubiquinone and reduce oxygen to water by bypassing the QoI-induced block in the electron transport chain allowing growth [7,9,10].
Field resistance to QoI fungicides in E. necator population was detected in USA, several European countries and Australia [11,12].It has been confirmed that the G143A mutation in cytb confers QoI resistance in E. necator [13,14].
The development of suitable monitoring techniques and effective anti-resistance strategies are crucial to maintain effectiveness of QoI fungicides.The objective of the present work is to determine the baseline sensitiveity and resistance of E. necator genetic groups to azoxystrobin fungicides at molecular level.

Fungal Isolates
The fungal isolates used in this study were obtained from the characterized collection at University of Bari [1].In vitro grapevine leaves production and maintenance of the fungal isolates were carried out as described by Miazzi et al. (1997) [15].
In vitro produced grape leaves of cv."Baresana" were used to assess the sensitivity of E. necator isolates to fungicides [2].Leaves were standardized in age and size to minimize any possible influence of such factors on the growth of E. necator colonies.
Application of fungicides was carried out by dipping leaves in fungicide suspensions at appropriate concentration containing 0.05% tween 20 for 1 min under gentle shaking.Control experiments were conducted by immersing leaves in sterile distilled water containing 0.05% Tween 20.Leaves were then placed in 55-mm-diam Petri dishes containing 10 ml of B0/2 substrate as described by Miazzi et al. (1997) [15].Petri dishes were left closed in a laminar flow cabinet for 8 h before inoculation.Each leaf was then inoculated at single point with about 15 -20 conidia under a stereomicroscope at 50× magnification.Inoculated leaves were then kept in a growth chamber at 21˚C ± 1˚C and exposed, 16 hours per day, to the light produced by a combination of 3 Osram L36W Cool White lamps and 3 Silvanya Grolux F36W lamps.
After two weeks, diameter of fungal colonies was measured with the aid of stereomicroscope.The effective concentration at 50% (EC 50 ) was calculated for individual isolates.Resistance factor (RF) was calculated according to the formula RF = EC 50 for the resistant isolate/ EC 50 of sensitive isolates.

Molecular Biology Assay
The primers RSCBF1 and RSCBR2, designed by Ishii et al. (2001) [16] on the ground of highly conserved regions of cytb gene in fungi and proved specific for the cytb gene in Sphaeroteca fusca, were used.
DNA was extracted from mycelium and conidia with InstaGene Matrix (Bio-Rad Laboratories, USA).PCR was performed in 25 l reaction mixtures containing 50 ng of total DNA, 2 mM MgCl 2 , 200 M each dNTP (Promega, Madison, WI, USA); 10 mM Tris-HCl, pH 8.3; 50 mM KCl; 0.1% gelatin, and 1 U of Red Taq DNA polymerase (Sigma, St Louis, Missouri, USA).PCR reactions were performed in a thermal cycler (Gene Amp PCR System 9700; Perkin-Elmer, Norwalk, USA) programmed as follow: 4 min at 94˚C, followed by 40 cycles of 30 sec at 94˚C, 1 min at 52˚C, 1.5 min at 72˚C, and a final extension stage of 7 min at 72˚C [16].PCR products were separated on 2% agarose gel (Bio-Rad Laboratories, USA) in 0.5× TBE buffer (45 mM Tris-borate, 1 mM Na-EDTA; pH 8) at 110 V for 120 min (Sub-Cell TM, Bio-Rad Laboratories, USA), The gal was stained with 1 μg•ml −1 ethidium bromide for 10 min and the bands were recovered by mechanical excision of small gel plugs.DNA fragments were eluted, purified using the Qiaex II Gel Extraction Kit (QIAGEN, Germany) and sequenced by MWG Biotech (Italy).

Generation of Azoxystrobin Resistant Putative Mutants
Mono-conidial cultures of the G82 isolate, were grown in vitro grapevine leaves treated with azoxystrobin.At 1month intervals, colonies were sub-cultured on leaves treated with increasing concentrations (0.1, 1, 5, 10 and 20 μg•ml −1 ) of the fungicide.After that cultures were maintained on leaves treated with 10 or 20 μg•ml −1 azoxystrobin.

Results
Twenty E. necator isolates representative of the tow genetic groups (  1).The StI and StII amplicons were sequenced and analyzed with FASTA sequences in Organelles Library of EBI GenBank.Our results showed that StI, but not StII, had a high similarity with the sequences of the mitochondrial cytb of other fungi (e.g.Erysiphe graminis, Magnaporthe grisea, Venturia inaequalis, Podosphaera fusca, Saccharomyces cerevisiae).The similarity within the best 100 scores was 68% -87.7% identity and 69% -87.7% for the un-gapped alignment (Figure 2).
Attempts of obtaining laboratory mutants resistant to QoI-STAR fungicides were only partially successful, since only few mutants were obtained and usually they displayed a low resistance level.Based on information known in other fungi, mitochondrial resistance may be unstable, since mitochondrial genome is present at a relatively high number of copies, and it is very unlikely that all of them carry resistance mutations [17].
An in vitro technique allowed to establish baseline sensitivity of E. necator to azoxystrobin fungicide were optimized.No significant differences in response to the fungicides were observed among the tested isolates.Nevertheless, differences were detected between group A and group B isolates.The same behavior of the two groups was observed in early study in response to triadimenol (DMIs) Fungicide [2].
The higher sensitivity to azoxystrobin of group A isolates might play a role in their disappearance in vineyards from June onwards, when intensive fungicides treatments, including azoxystrobin, are routinely applied.Type A isolates are usually collected on typical flag shoots early in April, at the time of highest susceptibility of shoots to powdery mildew, corresponding to the phenological stage BBCH 13 -16 (i.e. three to six unfolded leaves) [17,18].At this time, type A isolates have a higher chance to colonise the forming buds in which they can remain until the next season.This was further confirmed by the fact that the incidence of flag shoots in a vineyard can be predicted by assessing the extent of bud infection in the preceding year [19].The overwintering mode of group A isolates determines their prevalent asexual reproduction.This seems to be confirmed also by the low genotypic diversity found in this group, and by the unbalanced dis-tribution of the two mating type alleles that characterizes this group [1,20,21].If isolates of group A are present only at the beginning of the growing season, they have a reduced opportunity to be exposed to the fungicide pressure.This fact, coupled with the absence of the gene rearrangement consequent to sexual reproduction, would make group A isolates less likely to develop resistance.
In contrast, group B isolates develops later in the season and reproduce mostly sexually [22].Thus, recombinants would have a better opportunity of pyramiding mutations necessary to build the fungicides resistance that these isolates show, and that enables them to play a primary role in epidemics.
Further studies are required for a better understanding of E. necator resistance to azoxystrobin and for clarifying the complex interactions that this trait appears to have with population genetics of this pathogen.Ascertaining if the differential sensitivity of isolates of the two genetic groups extends also to other fungicide groups, identifying other factors that may favor one group over the other, and understanding how all these factors can affect the evolution of the epidemics, will be of great help for a more efficient disease control.

Figure 1 .
Figure 1.Electrophoretic profiles obtained by amplification of E. necator DNA with the primers RSCBF1-RSCBR2, and sequences of St I (A) and St II (B).

Figure 2 .
Figure 2. StI translation nucleic acid to amino acid (underlined) and multiple alignments with the cytochrome b gene fragments containing the point mutations responsible of resistance to QoI-STARs in other fungi (in bold, position 129 and 143).

Table 1 )
, were used to establish baseline sensitivity to azoxystrobin.EC 50 values ranged from less than 0.1 to 0.3 μg•ml −1 azoxystrobin.Colony growth was inhibited at 0.1 to 6 μg•ml −1 of the fungicide.Differences in EC 50 values among tested isolates were very low.MIC values showed by isolates belonging to group B were between 1 -6 μg•ml −1 azoxystrobin, were slightly higher than that of group A isolates.MIC values of group A isolates wer between 0.1 -1 μg•ml −1 azoxystrobin.It is obvious that in most cases, the highest MIC value for group A isolates corresponds to the lowest MIC value for group B isolates.DNA amplification with he primers RSCBF1 and t