Characterization of Monilinia species associated with brown rot in stone fruit in Brazil

Fungi of the Monilinia genus occur worldwide and affect a wide range of economically important stone fruits. Several Monilinia species are responsible for brown rot. Although this disease is common in Brazil, Monilinia sp. genetic variability in Brazilian orchards has generally been poorly characterized. The present study represents the first report on the genetic diversity of Monilinia sp. from Brazilian orchards. The genetic structure of the Brazilian population was also compared to isolates from other countries, together with some morphological characteristics and aggressiveness. Sixty‐one isolates belonging to the Monilinia genus were obtained from different orchards in Brazilian states. Ten Monilinia fructicola isolates from the United States and one isolate from a fruit imported into Brazil were also evaluated. Phylogenetic analysis of the ITS1‐5.8S‐ITS2 region (internal transcribed spacer) clustered most Brazilian and American isolates with M. fructicola authentic strains from Q‐Bank. Two isolates (one from an imported fruit) clustered as Monilinia laxa. The results revealed M. fructicola as the prevalent species associated with brown rot in Brazilian orchards. To evaluate the intraspecific diversity of M. fructicola and M. laxa, multigene sequence analysis was performed using ITS1‐5.8S‐ITS2 and TEF1 (elongation factor 1). Whilst TEF1 is the most phylogenetically informative gene for intraspecific studies of M. fructicola, RPB2 (RNA polymerase II gene) displayed low variation in intraspecific analysis, but was an informative locus for assigning isolates to M. fructicola or M. laxa species. The amova suggests that Brazilian isolates from the States of the main producing regions belong to a single genetic population, which is genetically distinct from the US (Californian) population of M. fructicola.     

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.     

Epidemiology of brown rot (Monilinia fructigena) on apple: infection of fruits by conidia

Effects of fruit maturity, wound age, temperature and the duration of wetness periods on infection of apple fruits by conidia of the brown rot fungus, Monilinia fructigena , were studied. Inoculation of fruits on potted apple trees and harvested mature fruits showed that wounding was essential for infection by M. fructigena . On potted trees, there was a significant difference between the susceptibility of cvs Cox and Gala and this difference depended on wound age. The incidence of brown rot was affected greatly by fruit maturity and wound age. Wounds on younger fruits were more resistant to infection than those on older fruits, whilst the older the wound, the more resistant it was to infection. Furthermore, the degree of wound age-related resistance was greater on younger fruits than on older fruits. These relationships were well described by regression models. The effect of the duration of wetness periods was very small: increasing the duration of wetness periods reduced the incidence of brown rot on older wounds. For detached fruits, all those wounded were rotted after inoculation, except for those in two treatments under 20°C on fruits with wounds which were 8 days old. The incubation period of the fungus was generally very short. Wound age was the single most important factor influencing the length of the incubation period; the incubation period increased as wound age increased.     

Frequency of brown rot fungi on blossoms and fruit in stone fruit orchards in Greece

Brown rot is a devastating disease of stone fruits caused by Monilinia spp. This study was conducted to investigate the disease aetiology on blossoms and fruit in peach, apricot, sweet cherry and plum orchards, in Greece. In total, 1433 isolates obtained from orchards located in the main stone fruit production regions of Greece were identified to species based on the presence/size of a cyt b intron. Monilinia laxa and M. fructicola were detected at frequencies of 59 and 41%, respectively, while M. fructigena was absent. Monilinia fructicola was more common on fruit whereas M. laxa occurred in similar frequency on blossoms and fruit. Monilinia laxa was replaced by M. fructicola in fruit infections of peach in both regions investigated and in fruit infections of plum in the Imathia region. Assessments of aggressiveness of 30 isolates of both species on the petals and fruits of the hosts showed that M. fructicola isolates were more aggressive. This suggests that the predominance of M. laxa on the blossoms cannot be explained by higher aggressiveness. Measurements of the effect of temperature on mycelial growth showed that M. laxa isolates had a higher growth rate than M. fructicola at the lowest temperature tested of 5°C, whereas M. fructicola isolates showed higher growth rates at higher temperatures. The observed high frequency of M. fructicola in Greece represents a major threat for stone fruit production. Furthermore, the information obtained about delineation of species and plant organ preference could be useful for the implementation of disease management strategies.     

Characteristic of Monilinia spp. fungi causing brown rot of pome and stone fruits in Poland

Brown rot, caused by fungi belonging to the genus Monilinia, is one of the most important diseases of stone and pome trees in the world. During the summers of 2010 and 2011, a total of 670 Monilinia spp. isolates were obtained from infected fruits. They were collected from different commercial stone and pome fruit orchards, located in northern, southern and central Poland. All isolates were identified using multiplex PCR. Twenty isolates obtained from plum, peach and apple fruits were identified as M. polystroma and 5 isolates from plums as M. fructicola. The remaining isolates were identified as M. fructigena or M. laxa. The identification of the isolates was also confirmed on the basis of growth characteristics in culture according to the EPPO standard PM 7/18. A comparison of morphological features of four Monilinia spp. growing on two selective growth media, APDA-F500 and CHA, indicated significant differences between these species. In artificial inoculation of fruits, all the examined Monilinia spp. isolates were pathogenic. The species affiliation of M. polystroma and M. fructicola isolates collected from orchards in Poland was confirmed on the base of phylogenetic and sequence analysis of the internal transcribed spacer (ITS1/5.8S rDNA/ITS2) region of ribosomal DNA.     

桃褐腐病的发生和防治

链核盘菌（Monilinia spp.）所致的褐腐病是核果类果树的主要病害之一。我国16省（区）记载发生此病。本文综述了前人关于病原菌鉴定、分布、侵染特点和病害循环等方面的研究进展,对褐腐病防治策略和防治中的难点进行了讨论。     

核果类果树褐腐病在重庆的发生与防治初报

近年随着乡村产业调整和果业发展, 褐腐病在我国的发生呈加重趋势。本文对重庆地区核果类果树褐腐病的发生状况做了调查分析, 在室内通过平板及离体果实试验, 筛选拮抗菌和化学药剂, 为田间防治实践提供科学依据。主要结果如下:桃褐腐病在重庆普遍发生, 核果类果树均易感, 病原菌经鉴定为果生链核盘菌Monilinia fructicola; 系统调查初步分析发现, 降雨时间与李褐腐病发生发展关系最密切, 其与病情指数增幅的相关系数R=0.94(P<0.05); 平板对峙和离体桃果筛选, 获得2株有生防潜力的放线菌YLS5-2和YYDB3-1, 二者的抑菌率分别为91.3%和84.5%, 相对防效分别为65.1%和67.1%, 可能具有较好的应用前景; 平板毒力测定和离体桃果控病试验, 效果最优的化学药剂分别为戊唑醇、苯甲·丙环唑, 其中戊唑醇在推荐浓度处理96 h其离体果实防效仍维持在100%水平。     

Field application of Bacillus subtilis and Aureobasidium pullulans to reduce Monilinia laxa post-harvest rot on cherry

European Journal of Plant Pathology - Brown rot caused by Monilinia spp. is one of the most important diseases in stone fruits worldwide. Latent infections of fruit by the pathogen often manifest...     

Quantitative relationships between different injury factors and development of brown rot caused by Monilinia fructigena in integrated and organic apple orchards

In a 4-year study, the incidence of various types of injuries (caused by insects, birds, growth cracks, mechanical wounding, and other, unidentified factors) was assessed in relation to brown rot development (caused by Monilinia fructigena) on fruit of three apple cultivars (Prima, Jonathan, and Mutsu) in integrated and organic blocks of two apple orchards in Hungary. In addition, populations of male codling moths (Cydia pomonella) were monitored with pheromone traps season-long in both management systems. On average, injury incidence on fruit at harvest was 6.1 and 19.2% in the integrated and organic treatments, respectively. Insect injury, which was caused primarily by C. pomonella, had the highest incidence among the five injury types, accounting for 79.4% of the total injury by harvest in the organic blocks and 36.6% in the integrated blocks. Levels of all other injury types remained close to zero during most of the season, but the incidence of bird injury and growth cracks increased markedly in the final 3 to 5 weeks before harvest in both production systems. Brown rot developed more slowly and reached a lower incidence in the integrated (6.4% final incidence on average) compared with the organic blocks (20.1% average incidence). In addition, the disease developed later but attained higher levels as the cultivar ripening season increased from early-maturing Prima to late-maturing Mutsu. Overall, 94.3 to 98.7% of all injured fruit were also infected by M. fructigena, whereas the incidence of brown-rotted fruit without visible injury was very low (0.8 to 1.6%). Correlation coefficients (on a per plot basis) and association indices (on a per-fruit basis) were calculated between brown rot and the various injury types for two selected assessment dates 4 weeks preharvest and at harvest. At both dates, the strongest significant (P < 0.05) relationships were observed between brown rot and insect injury and between brown rot and the cumulative number of trapped C. pomonella. At the harvest assessment, two additional significant correlations were between brown rot and bird injury and between brown rot and growth cracks. In every case, correlation coefficients were larger in organic than in integrated blocks. Although it is well established that brown rot in pome fruits is closely associated with fruit injuries, this is the first study to provide season-long progress data on different injury types and quantitative analyses of their relative importance at different times in the growing season and across two distinct management systems.     

Characterization of the cytochrome b (cyt b) gene from Monilinia species causing brown rot of stone and pome fruit and its significance in the development of QoI resistance

BACKGROUND: Quinone outside inhibitor (QoI) resistance as a consequence of point mutations in the cytochrome b (cyt b) gene has been reported in numerous plant pathogenic fungi. To examine the potential for QoI resistance development in those Monilinia species causing brown rot of stone and pome fruits [Monilinia fructicola (G Winter) Honey, M. laxa (Aderhold & Ruhland) Honey and M. fructigena (Aderhold & Ruhland) Honey], an examination was made of the sequence and exon/intron structure of their cyt b genes for the presence of any point mutations and/or introns commonly associated with resistance to QoIs in fungal plant pathogens. RESULTS: None of the point mutations typically linked to QoI resistance was present in any of the Monilinia isolates examined. Furthermore, the cyt b genes from M. fructicola and M. laxa, but not M. fructigena, possessed a group‐I‐like intron directly after codon 143. Based on the results obtained, a simple PCR assay using a single primer pair was developed, allowing discrimination between the three Monilinia species without the need for culturing. CONCLUSIONS: Results suggest that resistance to QoI fungicides based on the G143A mutation is not likely to occur in M. fructicola or M. laxa. Conversely, M. fructigena may be at higher risk for developing QoI resistance owing to the absence of a G143‐associated intron. Copyright © 2010 Society of Chemical Industry     

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。     

Modelling the effect of cuticular crack surface area and inoculum density on the probability of nectarine fruit infection by Monilinia laxa

The effects of cuticular crack surface area and inoculum density on the infection of nectarine fruits by conidia of Monilinia laxa were studied using artificial inoculations with conidial suspensions and dry airborne conidia during the 2004 and 2005 seasons, respectively. Additionally, the effect of ambient humidity on fruit infection was evaluated in the 2005 experiment. An exploratory analysis indicated that (i) ambient humidity did not significantly explain the observed variability of data, but that (ii) the incidence of fruit infection increased both with increasing inoculum density and increasing surface area of cuticular cracks. The product of these two variables represented the inoculum dose in the cracks, and was used as a predictor of fruit infection in the model. Natural infection in the orchard was observed to increase throughout the season in both 2004 and 2005. The relationship between the probability of fruit infection by M. laxa and the artificially inoculated dose in the cuticular cracks was well described by a logistic regression model once natural inoculum density was taken into account (pseudo R² = 65%). This function could be helpful for estimating the risk of fruit infection at harvest based on fruit size and natural inoculum density.     

A real‐time (TaqMan) PCR assay to differentiate Monilinia fructicola from other brown rot fungi of fruit crops

To prevent the entry and spread of the brown rot fungus Monilinia fructicola in Europe, a fast and reliable method for detection of this organism is essential. In this study, an automated DNA extraction method combined with a multiplex real‐time PCR based on TaqMan chemistry was developed for fast, convenient and reliable detection of both the EU quarantine organism Monilinia fructicola and the three other brown rot fungi M. fructigena, M. laxa and Monilia polystroma. Using the internal transcribed spacer (ITS) region of the nuclear ribosomal RNA gene repeat, a Monilinia genus‐specific primer pair and two differently labelled fluorogenic probes specific for M. fructicola and the group M. fructigena/M. laxa/Monilia polystroma were developed. The analytical specificity of the assay was assessed by testing 33 isolates of the four brown rot fungi and 13 isolates of related fungal species or other fungal species that can be present on stone and pome fruit. No cross‐reactions were observed. The assay was found to have a detection limit of 0·6 pg of DNA, corresponding to 27 haploid genomes or four conidia. Comparison of a manual DNA isolation followed by a conventional PCR with an automated DNA isolation combined with the presently developed real‐time PCR showed that the latter method gave improved results when tested with 72 naturally infected stone fruit samples. The detection rate increased from 65 to 97%.     

Signals induced by exogenous nitric oxide and their role in controlling brown rot disease caused by <Emphasis Type="Italic">Monilinia fructicola</Emphasis> in postharvest peach fruit

Recent evidence suggests that nitric oxide (NO) signaling plays an important role in plant–pathogen interactions and that aconitase is a major target of NO. In the present study on the signaling role of NO in the elicitation of defense responses in peach fruit against Monilinia fructicola and subsequent effect on brown rot disease, 15 μM NO solution induced disease resistance in harvested peaches. As a potentiated elicitor, NO induced high levels of endogenous NO and superoxide (O₂ ^?), hydrogen peroxide (H₂O₂), and NADPH oxidase and Ca²⁺-ATPase activity in the fruit. Aconitase activity in peach fruit was inhibited by NO. Activity of partially purified aconitase was inhibited in vitro by sodium nitroprusside (SNP) and H₂O₂; however, the inhibition could be relieved by carboxy-2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (cPTIO) or catalase (CAT), indicating that the defense response and signals induced by NO transduction depend on aconitase and conditions leading to elevated levels of NO; otherwise, H₂O₂ would inactivate aconitase directly in fruit. Treatment with NO resulted in salicylic acid (SA) accumulating during storage. Higher levels of jasmonic acid (JA) were detected in NO-treated fruit 48 h after the treatment. But after NO was removed, the level of SA and JA were lower than in the control. The results suggest that exogenous NO enhances resistance of harvested peach fruit against the fungus by inducing signals such as endogenous NO, reactive oxygen species (ROS), SA and JA and by inhibiting aconitase activity.     

进口智利李子上核果褐腐病菌鉴定

从进口智利李子上分离到一种引起李子果实腐烂的病原真菌,接种李子能引起果实变褐和腐烂。该真菌菌丝在PDA培养基上的生长最适温度为25℃,产孢少,菌落初始为浅灰色,边缘浅裂状,多次传代后逐渐变为浅白色,边缘整齐。分生孢子无色,单孢,柠檬形或卵圆形,大小11．5（9．5～14．7）μm&#215;7．4（5．3～9．2）μm。ITS序列分析结果表明该分离物ITS区序列与GenBank中登录的16株舱laxa序列相似性分别为99．8％～100％,碱基的差异为0～1bp;与21株旭frueticola序列相似性为99．0％～99．2％,碱基的差异为4～5bp;与10株M.fructigena的序列相似性为97．5％～97．7％,碱基的差异为13～14bp。基于ITS序列的系统发育关系分析表明分离物sh675和GenBank中登录的16个M.laxa菌株属于同一个聚类群,相关的Mfructieola和M.fructigena属于另一个聚类群。M.laxa特异性引物ITS1Mix／ITS4Mlx能扩增分离物sh675的菌丝DNA,得到预期的356bp扩增产物。根据该真菌的菌落形态特征、分生孢子形态特征、ITS序列和PCR检测结果,将其鉴定为核果褐腐病菌（Monilinia laxa）。     

Factors influencing internal fruit rot of cucumber caused by Didymella bryoniae

Several factors influencing the incidence of internal fruit rot of cucumber caused byDidymella bryoniae were studied.Internal infection of fruits is achieved via the flower. However, in most cases the majority of the fruits escaped infection after flower inoculation. It took more than two days for the fungus to reach the fruit after infection of the style. A mechanical barrier was not detected in the fruit tip within three days after inoculation of the open flower.Inoculation of wilted flowers resulted in 60% less infection than inoculation of fresh flowers. Blossom excision reduced fruit infection with ca 75%. Growing plants under drought stress markedly increased the incidence of internal fruit rot.Neither the method of inoculation, nor the composition of the inoculum, nor the relative humidity influenced the incidence of internal fruit rot. Fruit thinning, duration of fruit growth, flowering period and the removal of parts of the flower had no effect either on fruit infection.Cultivars resistant to powdery mildew were also resistant to internal fruit infection. The resistance was associated with a long style and a short flowering period.Growing cultivars in which the flowers quickly fall away from the fruitlets or in which the flowers have no style may solve the problem of internal fruit rot in cucumber.Samenvatting Factoren die van invloed kunnen zijn op het ontstaan van inwendig vruchtrot van komkommer, veroorzaakt doorDidymella bryoniae, werden onderzocht.Inwendige vruchtinfectie vindt plaats via het bloempje. Bijna altijd ontsnapte echter het grootste deel van de vruchten aan een aantasting als de bloem werd geïnoculeerd. Het duurde meer dan twee dagen voordat de schimmel via de stijl de vrucht had geïnfecteerd. In de punt van de vrucht werd binnen drie dagen na inoculatie van de open bloem geen mechanische barrière gevonden.Na inoculatie van verwelkte bloemen kwam 60% minder aantasting voor dan na inoculatie van bloemen die pas open waren. Het verwijderen van het bloempje reduceerde de aantasting met ca. 75%. Het optreden van inwendig vruchtrot nam aanzienlijk toe door de planten onder droge omstandigheden te telen.Noch de methode van inoculatie, noch de samenstelling van het inoculum, noch de relatieve luchtvochtigheid beïnvloedden het optreden van inwendig vruchtrot. Vruchtdunning, duur van de vruchtgroei, bloeiduur en het verwijderen van delen van de bloem hadden ook geen effect op de aantasting.Cultivars die resistent waren tegen echte meeldauw vertoonden ook resistentie tegen inwendige vruchtaantasting. De resistentie was gecorreleerd met een lange stijl en met een korte bloeiduur.De teelt van cultivars waarvan de bloemdelen snel van de vruchtbeginsels afvallen, of waarvan de bloempjes geen stijl hebben, zou het probleem van inwendig vruchtrot bij komkommer kunnen oplossen.     

Factors influencing external fruit rot of cucumber caused by Didymella bryoniae

Several factors influencing the occurrence and extent of external fruit rot caused byDidymella bryoniae on cucumbers in the post harvest period were studied.The minimum, optimum and maximum temperatures for growth of the fungus on fruits were circa 10, 23 and 35°C, respectively. The influence of the temperature on the growth of the fungus in vitro and in vivo was about similar. The fitness of the fungus diminished by storing inoculated fruits at about the maximum temperature for growth of the fungus for one day, but this temperature influenced fruit quality negatively. Storing at 10 to 12°C is more advisable.Isolates ofD. bryoniae showed variation in virulence. There was a linear relationship between growth on fruits and growth in vitro of these isolates, but no correlation was found with disease incidence on plants.The degree of fruit rot was increased by more severe wounding, by storing in the dark instead of in the light and by higher nitrogen fertilization of the crop. Relative humidity during storage had no effect on fruit decay. It is very likely that the amount and composition of available nutrients for fungus growth determine the degree of rotting of the fruits.With the present cultivars, external fruit rot can be best controlled by reducing the changes of wounding in the pre- and post-harvest period.Samenvatting Verschillende factoren die van invloed kunnen zijn op het ontstaan en de mate van uitwendig vruchtrot op komkommers in de periode na de oogst, veroorzaakt doorDidymella bryoniae, zijn onderzocht.De minimum, optimum en maximum temperatuur voor de groei van de schimmel op vruchten waren respectievelijk circa 10, 23 en 35°C. De invloed van de temperatuur op de groei van de schimmel in vitro en in vivo was nagenoeg gelijk. Door geïnoculeerde vruchten een dag bij de maximum temperatuur voor de groei van de schimmel te bewaren, werd de groeikracht van de schimmel verminderd, maar de vruchtkwaliteit werd door deze temperatuur negatief beïnvloed. Het is raadzamer de vruchten bij 10–12°C te bewaren.Isolaten vanD. bryoniae vertoonden een variatie in virulentie. Tussen de groei van deze isolaten op vruchten en de groei in vitro bleek een lineair verband te bestaan, maar er bestond geen verband met de aantasting van planten.De mate van vruchtrot nam toe door de vruchten ernstiger te verwonden, ze in het donker in plaats van in het licht te bewaren en door een hogere stikstofbemesting tijdens de teelt. De relatieve luchtvochtigheid tijdens de bewaarperiode had geen effect op de vruchtaantasting. De hoeveelheden en de samenstelling van de voor de groei van de schimmel beschikbare voedingsstoffen bepalen zeer waarschijnlijk de mate van vruchtrot.Uitwendig vruchtrot kan bij de huidige cultivars nog het best worden tegengegaan door de mogelijkheden van verwonding, zowel in de periode voor als na de oogst, te verkleinen.     

甘肃省马铃薯束梗褐腐病病原鉴定及其生物学特性研究

对甘肃省马铃薯(Solanum tuberosum L.)束梗褐腐病病原进行了分离鉴定和生物学特性研究。经形态特征观察与rDNA-ITS序列分析, 将病原菌鉴定为细基束梗霉［Doratomyces stemonitis (Pers. ex Fr.) F.J. Morton & G. Smith］。生物学特性研究结果表明, 病菌菌丝生长最适温度为25～30 ℃, 最适pH为6, 病菌能利用多种碳源, 但以蔗糖最好, 氮源以甘氨酸最适, 光照对菌丝生长没有影响。分生孢子在5～40 ℃范围内均能萌发, 最适25 ℃, 最适pH为7, 分生孢子萌发需液态水, 湿度低于99%几乎不萌发, 马铃薯汁液和葡萄糖液对孢子萌发有较好的促进作用。本研究为马铃薯束梗褐腐病的防治提供了理论依据。