Spatial and temporal characteristics of dicarboximide-resistant strains of Monilinia fructicola and brown rot incidence in stone fruit

The spatial and temporal distribution of dicarboximide-resistant strains of Monilinia fructicola were investigated in six peach and nectarine orchard blocks in 1987–89 using a dispersion index (Lloyd's Patchiness Index, LPI), and spatial, temporal and spatio-temporal autocorrelation analyses (Moran's Coefficient, I ). The LPI values indicated that resistant strains were aggregated in all blocks in all years. Spatial correlations were not significant beyond one quadrat for any spatial proximity pattern in five of six blocks. Thus the spread of resistant strains was mostly restricted to the vicinity of the original focus. An absence of significant temporal correlation between years in five of the six blocks indicated poor persistence of resistant strains at specific locations. Only one significant temporal correlation was detected at one block and this could have arisen by chance. Significant spatio-temporal correlation was not detected, suggesting that there was no focus expansion or carry-over of resistant strain inoculum from the previous sampling date. Spatial, temporal and spatio-temporal autocorrelation analyses were consistent with previously reported laboratory results that resistant strains had not acquired all the necessary characteristics to remain in, or dominate, field populations. The spatial pattern of brown rot incidence was investigated at one block in a separate study in 1988 and 1989. There were no significant spatial correlations for brown rot incidence in 1988 for any of the spatial proximity patterns analysed. In 1989, however, significant correlations indicated ellipsoid aggregates of brown rot orientated along the orchard rows.     

Persistence and fitness of carbendazim- and dicarboximide-resistant isolates of Monilinia fructicola (Wint.) Honey in flowers, shoots and fruit of stone fruit

Flowers on detached peach shoots and ripe fruit were inoculated under controlled conditions in order to estimate the competitive ability of Monilinia fructicola isolates sensitive and resistant to carbendazim and dicarboximide fungicides. Isolates varied considerably, but there was no consistent relationship between carbendazim resistance and competitive ability; there was, however, evidence of reduced competitive ability in the single dicarboximide-resistant and the dual dicarboximide/high carbendazim-resistant isolate examined. The ability to produce conidia on twig cankers inoculated in late spring 1989 was retained under field conditions by all sensitive and resistant isolates for at least 1 year. Dicarboximide-resistant isolates produced fewer conidia than the carbendazim-resistant and sensitive isolates on cankers. The production of conidia on mummified fruit inoculated in February 1990 decreased in the field during the winter and the following spring, but some conidia were produced by all isolates. Measures of pathogenicity, virulence and fitness for all isolates were similar to the original values after survival for 1 year. The evidence presented in this paper supports field observations that carbendazim-resistant isolates are likely to persist permanently in the M. fructicola population, whereas dicarboximide-resistant isolates are more likely to decline unless fungicide selection pressure is maintained.     

从进境美国樱桃中首次截获美澳型核果褐腐病菌

Monilinia fructicola是我国禁止进境的检疫性真菌有害生物。广州白云机场口岸从美国输华樱桃中截获可疑病果,病果表面有微小的圆形褐色病斑。保湿培养4d后,病斑迅速扩展至全果,并簇生绒状灰白至灰黄色菌落,导致果实腐烂。经对分离物进行形态学观察和ITS序列分析,将病菌鉴定为美澳型核果褐腐病菌(Monilinia fructicola(Winter)Honey),从进境美国樱桃大宗货物中截获该病菌在我国尚属首次。     

Discontinuance of tebuconazole in the field restores sensitivity of Monilinia fructicola in stone fruit orchards

Demethylation inhibitor (DMI) fungicides are used to control brown rot in stone fruit worldwide. However, their specific mode of action can select resistant isolates of Monilinia fructicola. Monilinia fructicola resistant to DMI fungicides are associated with a fitness cost in the absence of selective pressure, indicating that the sensitive population can be re-established when discontinuing the fungicide in the field. This work aimed to build up the sensitive population of M. fructicola after discontinuing the use of tebuconazole for successive crop seasons. The sensitivity of M. fructicola to tebuconazole was assessed in four commercial peach orchards in Paraná and São Paulo States from 2012/13 to 2015/16. Different fungicide programmes were used and DMI fungicides were discontinued from 2013/14. The sensitivity of M. fructicola to tebuconazole was assessed by a mycelial growth assay in vitro and by determining the frequency of the G461S mutation in the MfCYP51 gene. The isolates from Paraná had high sensitivity to the fungicide across all seasons and the frequency of the G461S mutation remained below 5%. The isolates from São Paulo were highly resistant in the 2012/13 season; however, there was a gradual decline until 2015/16. In addition, the G461S mutation frequency in Sao Paulo State was about 80% in the 2012/13 season, but reduced until it was completely undetectable in 2015/16. These results provide evidence that resistance can be managed in orchards with high selective pressure to tebuconazole after discontinuing the use of the fungicide for at least 3 years.     

Adaptation to fungicides in Monilinia fructicola isolates with different fungicide resistance phenotypes

The ability to develop fungicide resistance was assessed in Monilinia fructicola isolates with different fungicide sensitivity phenotypes by adapting mycelium and conidia to increasing concentrations of selective fungicides and UV mutagenesis. Results showed that adaptation to Quinone outside inhibitor (QoI) fungicide azoxystrobin and sterol demethylation inhibitor (DMI) fungicide propiconazole was more effective in conidial-transfer experiments compared to mycelial-transfer experiments. DMI-resistant (DMI-R) isolates adapted to significantly higher doses of azoxystrobin in both, mycelial- and conidial-transfer experiments compared to benzimidazole-resistant (BZI-R) and sensitive (S) isolates. Adaptation to propiconazole in conidial-transfer experiments was accelerated in BZI-R isolates when a stable, nonlethal dose of 50 microg/ml thiophanate-methyl was added to the selection medium. One of two azoxystrobin-resistant mutants from DMI-R isolates did not show any fitness penalties; the other isolate expired before further tests could be carried out. The viable mutant caused larger lesions on detached peach fruit sprayed with azoxystrobin compared to the parental isolate. The azoxystrobin sensitivity of the viable mutant returned to baseline levels after the mutant was transferred to unamended medium. However, azoxystrobin resistance recovered quicker in the mutant compared to the corresponding parental isolate after renewed subculturing on medium amended with 0.2 and 1 microg/ml azoxystrobin; only the mutant but not the parental isolate was able to adapt to 5 microg/ml azoxystrobin. In UV mutagenesis experiments, the DMI-R isolates produced significantly more mutants compared to S isolates. All of the UV-induced mutants showed stable fungicide resistance with little fitness penalty. This study indicates the potential for QoI fungicide resistance development in M. fructicola in the absence of a mutagen and provides evidence for increased mutability and predisposition to accelerated adaptation to azoxystrobin in M. fructicola isolates resistant to DMI fungicides.     

Studies on some characteristics of dicarboximide-resistant isolates of Botrytis cinerea from protected lettuce

Isolales of B. cinerea obtained directly from protected lettuce crops were either sensitive or moderately resistant to dicarboximides. Types with a high level of resistance to dicarboximides developed under laboratory conditions. Isolates with a high level of resistance grew poorly and sporulated feebly on cucumber cotyledons or lettuce leaves whereas growth and sporulation of moderately resistant isolates were only slightly poorer than of sensitive isolates. Moderately resistant isolates were stable in culture and there was no evidence that they produced mixtures of sensitive and resistant conidia. In polythene tunnel studies with mixed B. cineaea populations, in the absence of dicarboximides. no highly resistant isolates and only a small percentage of moderately resistant isolates were recovered.     

Species-Specific Detection of Monilinia fructicola from California Stone Fruits and Flowers

ABSTRACT A set of molecular diagnostics was developed for Monilinia fructicola, causal agent of brown rot of stone fruits, capable of sensitive detection of the pathogen in planta. Species-specific repetitive sequences were identified from a partial library of 312 recombinant clones hybridized with total DNA, followed by subsequent screening for specificity. One hundred isolates, comprising 12 fungal species common to California stone fruits, were surveyed for specificity. Three clones hybridized to 60 geographically diverse M. fructicola isolates (California, Michigan, Georgia, Oregon, and Australia) to the exclusion of all other fungi surveyed, including the closely related M. laxa (n = 12). Two clones were identical and of extrachromosomal origin (pMF73 and pMF150), whereas the third (pMF210) migrated with uncut DNA. The sensitivity of all three was comparable and capable of detecting 50 pg of fungal DNA in dot blot hybridizations. Six species-specific primer pair sets were designed. They maintained the same specificity patterns observed in the initial hybridization surveys and were sensitive enough to detect 50 fg of fungal DNA template, approximately equivalent to 10 spores. The species-specific clones were capable of detecting the pathogen in planta, specifically from infected plum flowers and nectarine fruit tissue, using both hybridization- and polymerase chain reaction-based methodologies.     

The benefits of combining elemental sulfur with a DMI fungicide to control Monilinia fructicola isolates resistant to propiconazole

BACKGROUND: Management of demethylation inhibitor (DMI) fungicide resistance in Monilinia fructicola (G. Winter) Honey is a priority in peach orchards of the southeastern United States, but DMI fungicides are still an important component of antiresistance strategies in view of the few effective alternatives. The goal of this study was to investigate potential benefits of a sulfur/propiconazole mixture for the control of propiconazole-resistant isolates.RESULTS: The mixture provided the best control for propiconazole-resistant isolates, regardless of protective or curative application timings, or the presence or absence of fruit injury. Propiconazole-resistant isolates developed disease on detached fruit after protective or curative applications of propiconazole or its mixture with sulfur, but protective applications of the mixture significantly reduced (P = 0.05) disease symptoms compared with the individual compounds. Additive to slightly synergistic effects were observed for the mixture in protective treatments of peaches inoculated with propiconazole-resistant isolates.CONCLUSION: The results suggest that the addition of elemental sulfur to a DMI fungicide is likely to be a relatively inexpensive means to improve brown rot control in peach production areas where reduced sensitivity to DMI fungicides is suspected but has not led to noticeable control failure.     

Molecular characterization of the two-component histidine kinase gene from Monilinia fructicola

Brown rot of stone fruit caused by Monilinia fructicola (G. Wint) Honey is one of the most common fungal diseases in California. In this study, two laboratory-induced iprodione-resistant (LIR) mutants of M. fructicola were characterized by osmotic sensitivity, virulence on prune and sequence of the two-component histidine kinase gene (Mfos-1). The LIR mutants showed more sensitivity to osmotic stress and lower virulence on prune than their wild-type parent. Analysis of deduced amino acid of Mfos-1 showed that this protein exhibited all the characteristic features of the two-component histidine kinase genes, including osmotic sensing domain, six 90-amino-acid repeat motifs (coiled coil region) and kinase core and response regulator domains. Comparison of DNA sequences of the Mfos-1 from LIR mutants and the wild-type sensitive (S) isolate showed that LIR mutants had single point mutations in the coiled coil region of Mfos-1.     

Field occurrence of vinclozolin resistance in Monilinia fructicola

Strains of Monilinia fructicola resistant to vinclozolin were isolated from fruit affected by brown rot from an orchard where the fungicide had been used over four seasons. Resistant isolates were pathogenic to peach fruit, and resistance of one isolate was confirmed following dipping of inoculated fruit in fungicide suspensions. In culture, one of four resistant isolates was identical in colony morphology and sporulation to sensitive isolates from different geographic areas. The other resistant isolates produced dark mycelium on PDA and were slower growing. Vinclozolin-resistant isolates were resistant in vitro to two other dicarboximide fungicides, iprodione and procymidone.     

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).     

Comparison of different PCR tests to identify Monilinia fructicola

Monilinia fructicola was until very recently a regulated pest in the European Union, and EU countries were requested to monitor its presence on their territories. As accredited laboratories should use validated tests, the mycological laboratory of CRA‐PAV carried out a validation process for the multiplex based PCR test (Coté et al., 2004 ), that is one of the most widely used tests for the identification of M. fructicola, although this test is not described in the EPPO diagnostic protocol PM 7/18 (2) because the validation data were lacking. The performance characteristics of this multiplex PCR test were established according to the EPPO Standard PM 7/98 (1) and the test was compared in a collaborative study with the end point PCR test (Ioos & Frey, 2000 ), considered as the ‘standard test’. The validation data were obtained using different isolates of M. fructicola, M. laxa, M. fructigena and Monilia polystroma, as well as different fruit tissues. Four series of the DNA target at different concentration, repeated three times, were analyzed in four Italian laboratories. The results showed that the multiplex PCR detection test (Coté et al., 2004 ) was fit for diagnostic purpose, although the analytical sensitivity was significantly lower compared to the conventional PCR ‘standard test’.     

Instability of Propiconazole Resistance and Fitness in Monilinia fructicola

ABSTRACT The fitness and the dynamics of demethylation inhibitor fungicide (DMI) sensitivity in isolates of Monilinia fructicola sensitive (no growth at 0.3 mg/liter propiconazole) and resistant (>/=50% relative growth at 0.3 mg/liter propiconazole) to propiconazole were investigated. Overall, there was no considerable compromise in the fitness of resistant isolates compared to sensitive isolates of M. fructicola at the time of collection. Resistant and sensitive isolates differed in their sensitivity to propiconazole (P < 0.001) and incubation period (P = 0.044), but not in latent period, growth rate, spore production, and spore germination frequency (P > 0.05). Consecutive transferring on potato dextrose agar had an impact on conidia production, conidial germination, and growth rate (P < 0.0001). Consecutive transferring also had an impact on propiconazole sensitivity in resistant isolates. In the resistant isolates, sensitivity to propiconazole increased (R(2) = 0.960, P = 0.0034) within the first eight transfers. Similarly, sensitivity to propiconazole increased by 273% over the course of 34 months in cold storage in propiconazole-resistant isolates. Our results show that propiconazole resistance is unstable in vitro and that standard subculturing and cold storage procedures impact propiconazole sensitivity of resistant isolates. The instability of propiconazole resistance in M. fructicola may have important implications for disease management in that a reversion to propiconazole sensitivity could potentially occur in the absence of DMI fungicide pressure in the field.     

Factors Affecting Latent Infection of Prune Fruit by Monilinia fructicola

ABSTRACT Experiments were conducted in three prune orchards in California. In each orchard, inoculations with Monilinia fructicola, the causal agent of brown rot of stone fruits, were performed on branches of trees at bloom and fruit developmental stages. Five inoculum concentrations were used in each inoculation. Six and four wetness durations were created for each inoculum concentration at bloom and fruit developmental stages, respectively. Fruit were harvested 3 weeks before commercial harvest. The overnight freezing incubation technique was used to promote sporulation and to determine incidence of latent infection (ILI) of fruit brown rot. No differences in ILI among locations were found. A seasonal pattern of bloom and fruit susceptibility to latent infection was determined. Susceptibility to latent infection at bloom stage was at a moderate level and increased to reach the highest level at pit hardening stage. Subsequently, fruit susceptibility to latent infection decreased, reaching the lowest level in early June at embryo growth stage. Thereafter, the susceptibility increased again with fruit development and maturity until harvest. Linear relationships between ILI and inoculum concentration were obtained for most combinations of growth stage and wetness duration. Incidence of latent infection increased linearly with increased wetness duration at bloom stage and increased exponentially with increased wetness duration at early and late fruit developmental stages. The optimum temperatures for latent infection at pit hardening stage ranged from 14 to 18 degrees C, but the effect of temperature on latent infection was reduced at resistant stages. The temperature range favorable to latent infection varied for different wetness durations.     

Population dynamics of dicarboximide-resistant strains of Botrytis cinerea on grapevine in New Zealand

Changes in the proportion of dicarboximide-resistant strains of Botrytis cinerea were monitored in two vineyard trials conducted during the period from October 1985 to November 1987. Dicarboximide resistance frequency in samples taken prior to flowering from wood (dead cane-ends, dead tendrils, old bunch remains) was high (66%) in the first trial, and low (12%) in the second trial. In treatments where resistance frequency was high on wood in spring, it decreased during the phase of disease establishment in new bunches irrespective of whether or not dicarboximide fungicides were applied. In the absence of dicarboximide applications, resistance frequency showed little change during the phase of disease development in bunches. The resistance frequency increased, however, if dicarboximide fungicides were applied, the extent of increase depending on the number and timing of applications and on disease severity at the time of application. Where resistance frequency was high on the wood at harvest, it decreased over the following winter but increased again in spring even though no dicarboximides had been applied. The best disease control in both trials was obtained with a programme in which dicarboximide fungicides were only applied during the period from veraison until harvest.     

Monilinia species identified on peach and nectarine in Croatia,with the first record of Monilinia fructicola

During 2012, an official survey was conducted for Monilinia species present on peach and nectarine in Croatia. In total, 169 Monilinia spp. isolates were collected from 24 commercial orchards and identified according to morphology in culture and PCR. Eighty of the isolates were identified as Monilinia laxa, 70 as M. fructigena and 19 as M. fructicola. M. fructicola was found only at one location in the Mediterranean part of the country, and this is the first record of this quarantine fungus in Croatia. PCR diagnostic tests using M. fructicola‐specific primer pair MO368‐5/MO368‐10R repeatedly gave false negatives for some isolates. PCR tests using primer pair ITS1‐Mfc1/ITS4‐Mfc1 amplified M. fructicola‐specific product in all isolates and was therefore shown to be more suitable for diagnostic purposes.     

基于环介导等温扩增技术快速检测美澳型核果褐腐病菌

本文建立了美澳型核果褐腐病菌(Monilinia fructicola)环介导等温扩增技术,并利用此技术开展了M.fructicola的检测和鉴定。采用M.fructicola SCAR分子标记的MO368特异片段为靶标序列,设计并筛选出M.fructicola的特异性LAMP引物,建立了该病菌快速诊断方法。试验结果表明,仅M.fructicola的菌株DNA扩增后呈天蓝色的阳性反应,而在其他真菌的供试菌株中均呈紫色的阴性反应。检测灵敏度可达到100 pg/μL。利用该方法对无锡机场截获的7份疑似李子褐腐病样本进行检测,结果有3份样品呈LAMP阳性反应。本文建立的M.fructicola LAMP检测方法具有灵敏度高、特异性好,简便易行等优点,为美澳型核果褐腐病菌的快速检测提供了一种新技术。     

Risk Analysis for Latent Infection of Prune by Monilinia fructicola in California

ABSTRACT The quantitative relationships between incidence of latent infection (ILI) of prune by Monilinia fructicola and wetness duration (WD) for different bloom and fruit developmental stages and different inoculum concentrations were obtained. Three levels of ILI were considered as criteria for low, moderate, and high risks of latent infection, respectively. Seasonal patterns of WD leading to different risk levels of latent infection were obtained for low (IP(L)) and high (IP(H)) inoculum potential conditions in orchards. Longer WD was needed at a resistant than at a susceptible fruit developmental stage to induce similar levels of latent infection. The curves of WD leading to different levels of ILI over the growing season (risky WD curves) were used in risk analysis for latent infection. Multi-year historical WD data from 10 prune-growing locations in California were compared with risky WD curves. The percentage of days (P) with WD leading to a certain risk level of latent infection was calculated for each month from historical weather data. Under the IP(L) condition, the P distributions for low risk of latent infection were higher in March and April than in May and were the lowest in June for most locations. Under the IP(H) condition, the number of days that WD leading to low risk of latent infection in May increased compared with those under the IP(L) condition. The risk analysis approach was evaluated by using separate experimental data as incidence of fruit brown rot obtained from different prune orchards over years. Consistency between predicted overall risk levels of latent infection and observed incidence of fruit brown rot was obtained. The results demonstrated the usefulness of the risk analysis in decision support system for disease management.