Vegetative compatibility groups and sexual reproduction among Spanish Monilinia fructicola isolates obtained from peach and nectarine orchards, but not Monilinia laxa

The frequency of occurrence of Monilinia fructicola in the Ebro Valley, Spain has increased since its first appearance in 2006, and M. fructicola has displaced Monilinia laxa, the native species which is the main cause of brown rot in peaches in this valley. In order to determine the characteristics that may be related to the displacement, we studied the capacity to generate new genotypic combinations of M. fructicola under laboratory conditions. The morphology and parasitic ability from ten field isolates of M. fructicola and M. laxa collected from three different orchards in the valley, and sampling from five different lesions were studied. Nitrate-non-utilising (nit) mutants were generated in order to test the isolates for vegetative compatibility which was done by assessing their colony growth when cultured singly or in pairs on media that contained different nitrogen sources. For the M. fructicola isolates, five vegetative compatibility groups (VCGs) were identified using the nit mutants and six VCGs were identified when they were grown on potato dextrose agar dishes. In all instances, the vegetatively compatible M. fructicola isolates came mainly from the same orchard. Only one VCG displays the same morphological and competition characteristics. No VCGs were identified among the M. laxa isolates. We did not find any apothecia of M. laxa and M. fructicola isolates in the soil of the three orchards, but we were able to produce apothecia of M. fructicola in the laboratory. Our finding of sexual reproduction and VCGs in the M. fructicola isolates suggests that the genetic variability of M. fructicola could be maintained by sexual and/or parasexual recombination. © 2014 The British Mycological Society

Sensitivity of Monilinia fructicola from Spanish peach orchards to thiophanate-methyl, iprodione, and cyproconazole fitness analysis and competitiveness

Benzimidazoles, dicarboximides and demethylation inhibitors are the main group of fungicides used to control brown rot in Spain. The causal agents of brown rot in peach (Prunus persica (L.) Batsch) orchards in Spain are Monilinia laxa M. fructigena, and M. fructicola. The dynamics of fungicide sensitivity and fitness of M. fructicola population, the most recent species causing brown rot in the Ebro Valley (Lleida, Spain), were characterized by determining their resistance, fitness, and in vitro competitiveness to thiophanate-methyl (TM), iprodione (I), and cyproconazole (CPZ) in field isolates recovered over the 2006–2010 period. We found that (a) more than 95 % of the M. fructicola isolates are high TM-resistant, (b) more than 50 % of the M. fructicola isolates are I-resistant and these frequencies of occurrence did not change during our 5-year survey, and (c) a few CPZ-resistant isolates have been also detected in population since 2008. We identified five different fungicide-resistant (R) and/or fungicide-sensitive (S) phenotypes and found that our study population contains multiple fungicide-resistant isolates. Moreover, these fungicide-resistant isolates display high parasitic fitness on fruit and flowers and high competitiveness. These findings suggest that the TM, I, and CPZ resistance of M. fructicola isolates could be contributing as another factor on changing the frequency of occurrence of the three Monilinia species in the Ebro Valley. © 2014, Koninklijke Nederlandse Planteziektenkundige Vereniging

The effect of fungicide resistance on the structure of Monilinia laxa populations in Spanish peach and nectarine orchards

Fungicide resistance has been proposed as one of the causes for changes in the frequency of occurrence of different fungal pathogen species. The purpose of this investigation was to study the dynamics of fungicide sensitivity, resistance, fitness, and competitiveness of Monilinia laxa field isolates in peach and nectarine orchards. The investigation comprised 788 isolates, which were randomly collected from 121 peach orchards in the Ebro Valley during the 2006 to 2010 growing seasons. Resistance to thiophanate-methyl, iprodione, and cyproconazole was determined by plating on potato dextrose agar amended with discriminatory concentrations of fungicides and determining EC50 values. Significant (P = 0.0476) time-dependent differences in the number of thiophanate-methyl-resistant isolates in the M. laxa population were found. The frequency of thiophanate-methyl-resistant isolates (EC50 range 104.0–137.5 μg ml−1) progressively increased since 2007. Although the number of iprodione -resistant isolates in the population did not change over time, two subpopulations of isolates showed iprodione reduced sensitivity (EC50 2.62–4.19 μg ml−1). TMRIR or TMRIS have become the dominant strains in the M. laxa population since 2007, and isolates with the TMRIS phenotype were the fittest and the most competitive phenotype in this population. No cyproconazole-resistant isolates were detected. These findings suggest that the current brown rot management strategies in the Ebro Valley should be revised. © 2016, Koninklijke Nederlandse Planteziektenkundige Vereniging

Role of gluconic acid and pH modulation in virulence of Monilinia fructicola on peach fruit

Colonization of nectarines and peaches fruit by Monilinia fructicola was accompanied by local acidification of the host tissue. The fungus acidified the host tissue in peaches and nectarines from pH 4.5 and 4.45, to pH 3.75 and 3.9, respectively. Analysis of the acidification process in colonized fruit and secondary inducing media showed that gluconic acid was the main organic acid accumulated at the infection site and under liquid-culture conditions. When comparing a nectarine cv. Big Top and peach cv. Plácido with differing sensitivities to M. fruticola, a 250% higher accumulation of gluconic acid was observed in the susceptible peach cultivar than in the less susceptible nectarine cultivar. Under liquid conditions, at pH 3.6-3.7, the relative expression of transcripts of mfpg 2 and mfpg3, encoding for two polygalacturonase genes of M. fructicola, increased 12-fold and 6-fold, respectively, suggesting the importance of acidification for the secretion of pathogenicity factors by M. fructicola. Our results indicate that ambient pH is a regulatory cue for processes linked to pathogenicity of postharvest pathogens, and that specific genes contributing to pathogenicity are expressed as a result of the environmental pH created by the pathogen. © 2013 Elsevier B.V

Occurrence of Monilinia laxa and M. fructigena after introduction of M. fructicola in peach orchards in Spain

Seventeen field surveys were done in four commercial orchards during six consecutive fruit-growing seasons from 2006 until 2011 in order to determine the current frequencies of occurrence of M. laxa, M. fructigena, and M. fructicola and their relative contributions to postharvest brown rot in peaches and nectarines in the Ebro Valley. The relative frequencies of occurrence of Monilinia spp. were determined on three sources of primary inoculum and on three sources of secondary inoculum. The major relative frequencies of Monilinia spp. were significantly recorded (P = 0.05) from mummified fruit on the trees (approx. 42 %) and 7-day-old harvested fruit with brown rot (32 %), followed by that recovered from mummified fruit on the orchard bed (14 %), pruned branches on the orchard bed (8 %) and latent infections of immature fruit (3 %). We found that (a) the relative frequency of M. fructicola has increased over the years to coexist on the same level as at the time M. laxa, (b) M. fructigena is no longer a cause of brown rot in harvested peaches, (c), a progressive reduction in the time of the first appearance of Monilinia airborne conidia (r = -0.30, P = 0.003), and the time of the first latent infection (r = -0.44, P = 0.0001) was detected along years after correlation analysis, and (d) these displacements are not associated with an increased incidence of brown rot disease. The M. fructicola increase was due to its significantly increased presence in 7-day-old harvested fruit with brown rot (r = 0.73, P = 0.0009), in latent infections of immature fruit (r = 0.68, P = 0.002), on pruned branches on the orchard bed (r = 0.56, P = 0.018), and on mummified fruit sampled on the trees (r = 0.53, P = 0.03). This progressive increase was accompanied by a progressive reduction in the relative frequency of occurrence of M. laxa in 7-day-old harvested fruit with brown rot (r = -0.55, P = 0.021) and M. fructigena on mummified fruit sampled on the trees (r = -0.51, P = 0.03). © 2013 KNPV