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.     

桃褐腐病菌(Monilinia fructicola)对3种杀菌剂的敏感性

采用生长速率法测定了采自北京平谷区3个桃园的125株桃褐腐病菌对甲基硫菌灵、戊唑醇和异菌脲3种杀菌剂的敏感性,发现甲基硫菌灵对桃褐腐病菌的EC50主要分布在1.0×10^-5～0.2μg/mL,戊唑醇对桃褐腐病菌的EC₅₀主要分布在0.006～0.022μg/mL之间。异菌脲对桃褐腐病菌的EC₅₀主要分布在0.15～0.55μg/mL之间。研究结果表明,北京地区的桃褐腐病菌对这3种杀菌剂都比较敏感,未产生明显的抗药群体。建立了褐腐病菌对异菌脲抗药性的敏感基线。而且,数据分析表明:甲基硫菌灵、戊唑醇和异菌脲之间均不存在交互抗性。     

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.     

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’.     

An intron in the cytochrome b gene of Monilinia fructicola mitigates the risk of resistance development to QoI fungicides

BACKGROUND: The cytochrome b (Cyt b) gene is a key genetic determinant for quinone outside inhibitor (QoI) fungicide resistance in plant pathogenic fungi. A mutation at amino acid position G143 can cause qualitative resistance unless it is part of the recognition site for a self‐splicing intron. The objective of this study was to clone and sequence the Cyt b gene from Monilinia fructicola (Wint.) Honey, the causal agent of brown rot of stone fruits, and to assess the risk for the development of a mutation at position 143. RESULTS: The Cyt b gene of M. fructicola was 11 927 bp in size and contained seven introns located at cDNA positions (5′–3′) 204, 395, 430, 491, 507, 780 and 812 with sizes of 1592, 1318, 1166, 1252, 1065, 2131 and 2227 bp respectively. Sequence analysis revealed that the above‐mentioned 1166 bp intron, a self‐splicing group I intron, was located just downstream of the G143 codon. The Cyt b gene region covering the G143 location and the adjacent 1166 bp intron was PCR amplified and sequenced from Chinese and US isolates, indicating that the intron may be omnipresent in M. fructicola. CONCLUSION: This is the first complete Cyt b gene sequence published for M. fructicola or any other Monilinia species, forming the basis for molecular analysis of QoI fungicide resistance. Sequence analysis revealed that the G143A mutation responsible for high levels of QoI fungicide resistance in many plant pathogenic fungi may not develop in M. fructicola unless genotypes emerge that lack the 1166 bp intron. Copyright © 2010 Society of Chemical Industry     

桃褐腐病菌对多菌灵抗性的AS-PCR检测技术

由于杀菌剂的长期大量使用, 植物病原菌对杀菌剂抗性问题日趋突出, 严重影响病害防治的效果?褐腐病是一种在全世界范围内广泛发生和流行的重要果树病害?本文以桃褐腐病菌Monilinia fructicola为例, 基于已知的多菌灵抗性机理, 即靶标β微管蛋白基因TUB2的点突变E198A, 建立了一种桃褐腐病菌对多菌灵抗性的等位基因专化性PCR检测技术?结果表明, 碱基错配?内参引物?退火温度?dNTPs?Taq DNA聚合酶?专化性引物浓度及配比等因素均对检测效率有影响?经过对以上因素的优化, 并对优化后的检测技术进行专化性及灵敏度评价, 证实该检测技术能特异性地鉴别桃褐腐病菌M. fructicola对多菌灵抗性基因型E198A, 且检测灵敏度可达到4.342 pg/μL病菌DNA, 有望在实践中推广使用?     

Influence of storage approaches on instability of propiconazole resistance in Monilinia fructicola

BACKGROUND: The long‐term preservation of interesting phenotypes in plant pathogenic fungi allows for follow‐up studies in the future. Twelve storage approaches were investigated to determine their effects on instability of propiconazole resistance for three demethylation inhibitor (DMI) fungicide‐resistant and two DMI‐sensitive isolates of Monilinia fructicola. They included mycelium in PDA slants under mineral oil, in PDA plugs under 10% glycerol, on dried filter paper and conidia on silica gel, each stored for 36 weeks at 4, ? 20, and ? 80 °C. RESULTS: None of the storage approaches prevented the rapid decline of EC₅₀ values for propiconazole in the three resistant isolates, and no significant differences were found among storage approaches (P = 0.787) or between storage approaches and consecutive transfers (P = 0.053). Most of the decline in resistance occurred during the first 4 weeks of storage. The DMI resistance‐associated genetic element Mona, located in the immediate upstream region of the MfCYP51 gene, was still present in the three resistant isolates after 36 weeks of storage and weekly transfers. Furthermore, the Mona element and a portion of the MfCYP51 gene, which encodes the target enzyme for DMIs, did not reveal signs of DNA methylation. Resistance to propiconazole was partially regained in resistant isolates after two growth cycles on fresh peach fruit. CONCLUSIONS: Obtained data indicate that the decline of DMI resistance in M. fructicola cannot be prevented using commonly employed storage methods at various temperatures. The number of consecutive transfers and the storage duration prior to fungicide sensitivity tests in M. fructicola should be indicated in scientific papers. Copyright © 2011 Society of Chemical Industry     

The biological characteristics of dicarboximide-resistant isolates of Monilinia fructicola from New Zealand stone-fruit orchards

Dicarboximide-resistant isolates of Monilinia fructicola were detected in New Zealand stone fruit orchards in 1985. The EC₅₀ values of resistant isolates ranged from 3 to 217 mg a.i./l iprodione, compared with 0-3 to 0-7 mg a.i./l for sensitive isolates. The degree of dicarboximide resistance was maintained over nine generations in fruit tissues in most isolates, but in four isolates there was a significant decline. Three resistant and two sensitive isolates were selected for further study on agar and host tissues. Resistant isolates caused disease, grew and sporulated as well as sensitive isolates on flowers not amended with dicarboximide fungicides, but some were less fit on fruit. The pathogenicity and spore production of a resistant strain on flowers was reduced significantly by dicarboximide applied prior to pathogen inoculation. Fitness of isolates on flowers and on fruit was poorly correlated. Resistant isolates were significantly less competitive than sensitive isolates when mixed inocula were applied to flowers and fruit. The relative performance on flowers and fruit was not a reliable indicator of competitive ability.     

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.     

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.     

Quantification of allele E198A in beta-tubulin conferring benzimidazole resistance in Monilinia fructicola using real-time PCR

BACKGROUND: Thiophanate-methyl, a member of the benzimidazole class of fungicides, is used in California to control brown rot of stone fruit caused by Monilinia fructicola (G. Wint.) Honey. The goal of this study was to develop a real-time polymerase chain reaction (PCR) assay as an efficient method to quantify the E198A allele of beta-tubulin that confers benzimidazole resistance. RESULTS: Using the real-time PCR assay, the frequency of allele E198A (FEA) in a population was determined from the quantities of DNA amplified with the E198A allele-specific primer pair HRF/HRR and the M. fructicola-specific primer pair MfF6/MfR6. The average proportions of highly resistant isolates determined with the conventional fungicide sensitivity method were within the range of average FEA values determined with the real-time PCR assay. We also determined the FEAs of M. fructicola populations sampled from 21 stone fruit orchards in California. Only one orchard showed a high FEA over 0.20, seven orchards had values between 0.01 and 0.1, and 13 orchards had values less than 0.01. CONCLUSION: The real-time PCR assay developed in this study provides a potentially useful tool to efficiently quantify benzimidazole resistance for large M. fructicola populations.     

Evaluation of Erysiphe graminis f sp tritici field isolates for resistance to strobilurin fungicides with different SNP detection systems

A single nucleotide polymorphism (snp) in the cytochrome b gene confers resistance to strobilurin fungicides in Erysiphe graminis DC fsp tritici Marchal. On the basis of this point mutation three different types of molecular markers have been developed. Cleaved amplified polymorphic sequences and allele-specific PCR were used to score resistant and sensitive isolates from specifically selected regional populations across Europe. The results of molecular tests were in total agreement with the resistance phenotypes revealed by in vivo tests. Serial dilutions of mixed samples (resistant/sensitive) delimited the detection for strobilurin-resistant alleles to a range of 10-50% for both marker classes. Due to these detection limits no mixture of mitochondria within individual isolates was found. Denaturing high performance chromatography was used to increase the detection sensitivity for the mutant allele. Although the detection limit was lowered to 5-10%, there was no evidence for the existence of mixed mitochondrial genotypes.     

桃褐腐病菌抗药性分子机理研究进展

桃褐腐病是严重危害桃生产的重要病害之一。在实际生产中,该病害的控制主要以化学防治为主。由于杀菌剂的大量使用,桃褐腐病菌已对几类常用的杀菌剂产生了不同程度的抗药性。本文就桃褐腐病菌(Moniliniaspp.)对生产上使用的几大类杀菌剂的抗药性情况及抗药性分子机制研究进展进行简单概述和分析,以供国内同行借鉴与参考。     

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.     

小麦纹枯病菌对三唑酮不同敏感性菌株的生物学特性及对不同杀菌剂的敏感性

为预测小麦纹枯病菌对三唑酮抗药性的发展趋势,采用菌丝生长速率法测定了2013年分离自河南省16个地市的98株菌株对三唑酮的敏感性,比较不同敏感性菌株的生物学性状差异,并分析了其中22株菌株对其它10种杀菌剂的敏感性。结果表明:小麦纹枯病菌群体中已出现对三唑酮敏感性下降的菌株,其中有90株菌对杀菌剂的敏感性属于正态分布,以其EC_(50)均值1.91μg/mL作为小麦纹枯病菌对三唑酮的敏感性基线。敏感菌株菌丝生长速度较快,可达8.50~12.86 mm/d,菌核产量较多,为35.80~42.90 mg/皿,生存适合度较高;敏感性下降及抗性菌株的致病性及菌核萌发等都未发生改变,但菌丝生长速率仅为5.36~10.02 mm/d,菌核产量为9.00~36.90 mg/皿,均下降明显,生存适合度降低;低抗菌株与敏感菌株在菌落形态上相同,但菌丝分枝数增多且分枝菌丝变短。三唑酮对小麦纹枯病菌的毒力较小,EC_(50)大于其它10种杀菌剂;戊唑醇、氟环唑、烯唑醇、咯菌腈和噻呋酰胺的毒力均较大,其EC_(50)≤0.10μg/mL;其余5种杀菌剂的EC_(50)在0.13~0.69μg/mL之间;小麦纹枯病菌对三唑酮的敏感性与其它10种杀菌剂间的决定系数为0.003~0.200,存在微弱的正相关性,表明在生产中这些杀菌剂可与三唑酮轮换或交替使用。     

Threshold Conditions That Lead Latent Infection to Prune Fruit Rot Caused by Monilinia fructicola

ABSTRACT Inoculations were performed six to eight times in each of 10 prune orchards located in nine counties of California. In each inoculation, branches that bore 40 to 60 blossoms or 30 to 40 fruit were inoculated with conidial suspensions of Monilinia fructicola. Three inoculum concentrations and 14 to 16 h of humidity were used for each inoculation. All inoculated fruit were maintained on trees and harvested separately 2 weeks before commercial harvest. The incidence of latent infection (ILI) and percentage of branches with fruit rot (PBFR) were determined for each inoculation in each orchard. As the ILI increased, the PBFR also increased linearly. Five conditions that lead latent infection to fruit rot include (i) latent infection level; (ii) fruit developmental stage; (iii) inoculum concentration; (iv) total hours of relative humidity greater than 90% (hRH); and (v) total hours of dew period (hDEW) from mid-July to mid-August. Three levels of PBFR, 1, 5, and 10% were assigned, and threshold conditions that lead to these levels were determined based on the experimental results. The relative probabilities that lead latent infection to fruit rot (r_PBFR) at different fruit developmental stages were calculated. A preliminary decision support model to guide fungicide application was developed based on the above results. One of the four recommendations, safe, wait, check historical weather as a reference, and apply a fungicide immediately, could be provided based on the level of latent infection and the decision process developed through this study.     

Induction, Regulation, and Role in Pathogenesis of Appressoria in Monilinia fructicola

ABSTRACT Monilinia fructicola, which causes brown rot in stone fruit, forms appressoria on plant and artificial surfaces. On nectarine, the frequency of appressoria produced by conidial germlings depends to a large degree on the stage of fruit development, with numerous appressoria formed on immature (stage II) nectarine fruit, and no appressoria observed on fully mature fruit (late stage III). On polystyrene surfaces, appressorium formation was increased from <10% of germinated conidia to >95% of germinated conidia when the conidia were washed to remove residual nutrients and self-inhibitors. M. fructicola appressorium formation also appears to be regulated by the topography of the plant surface. On fruit, appressoria formed on stomatal guard cell lips, on the grooves of lateral cells adjacent to stomata or between two epidermal cells, and on the convex surfaces of epidermal cells. Pharmacological effectors indicate that cyclic AMP-, MAP kinase-, and calcium/calmodulin-dependent signaling pathways are involved in the induction and development of appressoria. KN-93, an inhibitor of calmodulin-dependent protein kinase II, did not inhibit conidial germination but did inhibit appressorium formation and brown rot development on flower petals, suggesting that appressoria are required for full symptom development on Prunus spp. petals.     

Relative parasitic fitness of isolates of Pyricularia oryzae Cav. with different sensitivities to fungicides

The relative parasitic fitness of isolates of Pyricularia oryzae with different sensitivities to isoprothiolane (di-isopropyl 1,3-dithiolan-2-ylidenemalonate) and IBP (S-benzyl O, O-di-isopropyl phosphorothioate) was studied in the absence of fungicides. Four field isolates (S-1,S-2,MR-1 and MR-2) and two in-vitro mutants (Rvt-1 and Rvt-2) were used for disease epidemics. S-l and S-2 were wild types; MR-1 and MR-2 were sensitive to isoprothiolane and moderately resistant to IBP, and Rvt-1 and Rvt-2 were resistant to both fungicides. Twelve epidemics were made by inoculating rice seedlings with mixed conidial suspensions of two isolates or mutants. The value of relative parasitic fitness (W = 0-1.0) was calculated for each isolate and epidemic. S-l and S-2 were stronger (W = 1.0) than MR-2, Rvt-1 and Rvt-2; but weaker (W = 0.75, 0.73, respectively) than MR-1. MR-1 was strongest among all isolates and mutants used. MR-2 was slightly weaker (W = 0.9-1.0) than S-l and S-2, but stronger than Rvt-1 and Rvt-2. Rvt-1 and Rvt-2 had smaller values of W, ranging from 0.25-0.58, in the epidemics with each field isolate. These results suggest that the proportions of in-vitro mutants do not increase unless intensive selection pressure is given, and would be expected to decrease rapidly after the selection pressure is removed. Isolates moderately resistant to IBP, such as MR-1 and MR-2, however, had high values of W, suggesting that they would increase or would not decrease rapidly in the absence of selection pressure. These results may well explain why isolates highly resistant to isoprothiolane and IBP have seldom been found, and why a large number of isolates moderately resistant to IBP but sensitive to isoprothiolane have been observed in the field.