Goran Delibašić

Genus Monilinia on pome and stone fruit species.

Different species of the genus Monilinia are common plant pathogens that endanger pome and stone fruit production worldwide. In Serbia, two species of this genus are widely distributed - M. laxa and M. fructigena, while M. fructicola, which is officially on the A2 EPPO List of quarantine pest organisms in Europe and on the 1A part I List of quarantine pest organisms in Serbia, has so far been detected only on stored apple and nectarine fruits. The most important control measures against these pathogens include chemical control in combination with adequate cultural practices, particularly under favourable conditions for disease development. Concerning that species of this genus can cause significant economic losses, knowledge of the pathogen biology, disease epidemiology and pathogen-host interactions is a necessary prerequisite for stable and profitable production of pome and stone fruits. [Projekat Ministarstva nauke Republike Srbije, br. III46008 and br. III43001]

First report of brown rot caused by Monilinia fructicola on nectarine in Serbia.

In August 2011, nectarine (Prunus persica (L.) Batsch var. nucipersica (Suckow) C. K. Schneid) fruit originated from Oplenac region with symptoms of fruit rot was collected at a green market in Belgrade. Fruit had large, brown, sunken lesions covered with grayish brown tufts. Symptoms resembled those caused by species of Monilinia including M. laxa, M. fructigena, or M. fructicola (2). In order to isolate the causal organism, small superficial fragments of pericarp were superficially disinfected with commercial bleach and placed on potato dextrose agar (PDA). The majority (32 out of 33) isolates formed rosetted non-sporulating colonies with lobed margins resembling those of M. laxa. However, one isolate (Npgm) produced an abundant, grayish-white colony with even margins and concentric rings of sporogenous mycelium, resembling those described for M. fructicola (2). Conidia were one-celled, hyaline, ellipsoid to lemon shaped, 7.38 to 14.76 × 4.92 to 9.84 μm, and borne in branched monilioid chains. The average daily growth on PDA at 24°C was 10.9 mm. A single-spore isolate of Npgm was identified as M. fructicola based on the morphology of colony and conidia, temperature requirements, and growth rate (2). Morphological identification was confirmed by an amplified product of 535 bp using genomic DNA extracted from the mycelium of pure culture and species-specific PCR for the detection of M. fructicola (2). The ribosomal internal transcribed spacer (ITS) region of rDNA of Npgm was amplified and sequenced using primers ITS1/ITS4. Sequence analysis of ITS region revealed 100% nucleotide identity between the isolate Npgm (GenBank Accession No. JX127303) and 17 isolates of M. fructicola from different parts of the world, including four from Europe (FJ411109, FJ411110, GU967379, JN176564). Pathogenicity of the isolate Npgm was confirmed by inoculating five surface-disinfected mature nectarine and five apple fruits by placing a mycelial plug under the wounded skin of the fruit. Nectarine and apple fruits inoculated with sterile PDA plugs served as a negative controls. After a 3-day incubation at 22°C, inoculated sites developed brown lesions and the pathogen was succesfully reisolated. There were no symptoms on the control nectarine or apple fruits. M. fructicola is commonly present in Asia, North and South America, New Zealand, and Australia, while in the EPPO Region the pathogen is listed as an A2 quarantine organism (3). In Europe, the first discovery of M. fructicola was reported in France and since then, it has been found in Hungary, Switzerland, the Czech Republic, Spain, Slovenia, Italy, Austria, Poland, Romania, Germany, and Slovakia (1). Most recently, M. fructicola was found on stored apple fruits in Serbia (4). To our knowledge, this is the first report of M. fructicola decaying peach fruit in Serbia. These findings suggest that the pathogen is spreading on its principal host plants and causing substantial economic losses in the Serbian fruit production. References: (1) R. Baker et al. European Food Safety Authority. Online publication. www.efsa.europa.eu/efsajournal . EFSA J. 9:2119, 2011. (2) M. J. Côté. Plant Dis. 88:1219, 2004. (3) OEPP/EPPO. EPPO A2 list of pests recommended for regulation as quarantine pests. Version 2009-09. http://www.eppo.org/QUARANTINE/listA2.htm . (4). M. Vasic et al. Plant Dis. 96:456, 2012.

Monilinia spp. Causing Brown Rot of Stone Fruit in Serbia

Brown rot is one of the most important pre- and postharvest fungal diseases of stone fruit worldwide. In Serbia, where production of stone fruit is economically important, Monilinia laxa and M. fructigena are widely distributed. In surveys from 2011 to 2013, 288 isolates of Monilinia spp. were collected from 131 localities in 16 districts and from six hosts in Serbia. Using multiplex polymerase chain reaction, phylogenetic analysis, and morphological characterization, three species of Monilinia were identified as the causal agents of brown rot of stone fruit: M. laxa (89% of isolates), M. fructigena (3%), and M. fructicola (8%). In 2011, M. fructicola was reported for the first time on stone fruit in Serbia, with only one isolate detected. More isolates of M. fructicola were detected in 2012 (2 isolates) and 2013 (20 isolates). The presence of M. fructicola, as well as its increased frequency of detection during the survey, may indicate a change in the population structure of these pathogens, which could have an important impact on brown rot disease management in Serbia.

Toxicity of copper hydroxide, dithianon, fluazinam, tebuconazole and pyraclostrobin to Didymella applanata isolates from Serbia

A study of the in vitro sensitivity of 10 isolates of Didymella applanata to copper hydroxide, dithianon, fluazinam, tebuconazole and pyraclostrobin, was conducted. The isolates were derived from diseased raspberry canes sampled during 2013 at five localities in western part of Serbia, known as the main raspberry growing region of the country. Prior to sensitivity testing experimental conditions for radial growth assay were optimized. The results showed that the temperature of 22°C, oatmeal agar medium and 12/12 hrs light/ darkness light regimen provided the best conditions for sensitivity tests. Most of D. applanata isolates were sensitive to the tested fungicides. The narrowest range of EC50 values was recorded for tebuconazole (1.42–2.66 mg L−1). The widest range of EC50 values was obtained for pyraclostrobin, ranging from 0.17 mg L−1 to 55.33 mg L−1. The EC50 values for the studied isolates were 39.48–51.19 mg L−1 for copper hydroxide, 12.12–18.73 mg L−1 for dithianon and 5.72–42.56 mg L−1 for fluazinam. According to resistance factor values, all D. applanata isolates were sensitive to copper hydroxide, dithianon and tebuconazole. Among tested isolates, six were highly resistant to pyraclostrobin (RFs in the range of 207.1–325.5) and two moderately resistant to fluazinam (RFs were 3 and 7.4), respectively.

Molecular detection of Monilinia fructigena as causal agent of brown rot on quince

SUMMARy Species of the genus Monilinia are important causal agents of fruit rot on pome and stone fruits in Serbia. The pathogen is very harmful, especially on small properties and cottage plantations where intensive control measures are not applied. Quince is important host for the pathogens of this genus. During spring 2010, intensive occurrence of mummified fruits overwintering on branches of the quince was observed. The pathogen was isolated using standard phytopathological methods. Pathogenicity of eight obtained isolates was tested by artificial inoculation of injured apple fruits. Identification was performed according to pathogenic, morphological and ecological properties, and was confirmed by Multiplex polimeraze chain reaction, PCR. All the isolates studied caused brown rot on inoculated apple fruits. The isolates form light yellow colonies with lobate margins, with single-celled, transparent, elliptical or oval conidia in chains, regardless temperature or light presence. Sclerotia are observed in 14 days old cultures. The highest growth rate of most of the isolates is at 27°C and in dark. Based on studied pathogenic, morphological and ecological characteristics, it was found that the Monilinia fructigena is causal agent of brown rot of quince. Using specific primers (MO368-5, MO368-8R, MO368-10R, Laxa-R2) for detection of Monilinia species in Multiplex PCR reaction, the expected fragment 402 bp in size was amplified, which confirmed that the studied isolates belonged to the species M. fructigena.

Development of a thyme essential oil formulation and its effect on Monilinia ructigena

Antifungal activity of thyme essential oil against Monilinia fructigena and development of an effective and stable oil formulation for agricultural use were studied in this paper. Bioactivity of the oil itself and its developed formulation, an emulsifiable concentrate (EC), was tested in vitro and in vivo. In vitro experiments, using a slightly modified agar overlay technique, showed that the initially emulsified thyme essential oil, as well as the developed formulation, significantly inhibited mycelial growth of M. fructigena in vitro. Experiments in vivo, performed on inoculated apple fruits, revealed that the formulation successfully decreased oil evaporation from the treated area and provided a significant level of M. fructigena suppression, 64.7-72.1% compared to the control. To our knowledge, an EC formulation of thyme essential oil for agricultural uses had never been developed before. The presented results are initial findings and evaluation of product activity should be continued in the field to determine its efficacy and activity spectrum, and to estimate the economic aspect of its use. [Projekat Ministarstva nauke Republike Srbije, br. III 46008]

Fungicide sensitivity, growth rate, aggressiveness and frost hardiness of Monilinia fructicola and Monilinia laxa isolates

Monilinia fructicola, the most destructive pathogen of the genus Monilinia, has recently been introduced into Serbia and many other European countries. Since then, many studies have been conducted to evaluate the characteristics of Monilinia species that have a role in the establishment and survival of the pathogen in new areas. The present study assessed the capacity of M. fructicola to repress and replace Monilinia laxa in Serbia based on: fungicide sensitivity, growth rate and aggressiveness at different temperatures, as well as frost hardiness of the isolates of both species. The results showed that the isolates of M. fructicola, compared to M. laxa, were significantly less sensitive to the following fungicides: iprodione, tebucanozole, chlorothalonil, azoxystrobin, fluopyram, and boscalid. In addition, M. laxa isolates exhibited little variation in sensitivity to all of the tested fungicides, whereas M. fructicola isolates displayed a wide range of sensitivity. The temperature of 5°C favored M. laxa growth and aggressiveness, while at 30°C M. fructicola grew faster and had higher lesion expansion rate. These results support an assumption that M. fructicola will continue to spread in Serbian orchards in coming years, particularly on stone fruits harvested during hot summer weather.

Toxicity of mancozeb, chlorothalonil, captan, fluopyram, boscalid, and difenoconazole to Didymella applanata isolates from Serbia

Field isolates of Didymella applanata, the causal agent of spur blight of raspberry, were evaluated in vitro for their sensitivity to mancozeb, chlorothalonil, captan, fluopyram, boscalid and difenoconazole. A total of 10 isolates, collected during 2013 at five localities in the major raspberry growing region in Serbia, and characterized as copper hydroxide, dithianon, and tebuconazole (sensitive), pyraclostrobin (sensitive or highly resistant) and fluazinam (sensitive or moderately resistant), were used in this study. The EC50 values for the isolates ranged from 1.33 to 2.88 mg L−1 for mancozeb, from 3.18 to 6.65 mg L−1 for chlorothalonil, from 15.75 to 24.69 mg L−1 for captan and from 1.80 to 8.20 mg L−1 for fluopyram. The narrowest range of EC50 values was recorded for difenoconazole (0.23–0.49 mg L−1), whereas the widest range was obtained for boscalid (4.49–49.25 mg L−1). The calculated resistance factors showed that all D. applanata isolates were sensitive to mancozeb, chlorothalonil, captan, and difenoconazole. Four isolates were moderately resistant to boscalid, while three of them were also moderately resistant to fluopyram. This finding of moderately resistant isolates to these SDHI fungicides indicates a possible cross-resistance which should be clarified in further investigations.

Is low efficacy of fungicides always a consequence of fungicide resistance development in pathogen populations

Efficacy of four fungicides with different modes of action (vinclozolin, pyrimethanil, benomyl and fenhexamid) in control of B. cinerea in raspberry, was investigated in the paper. The trials were conducted at two localities in commercial raspberry plantations. In the case of unsatisfactory fungicide efficacy, qualitative and/or quantitative test of the susceptibility of the isolates to particular fungicide was performed, to determine whether the low efficacy is a consequence of resistance development in the pathogen population. At both localities, pyrimethanil and fenhexamid demonstrated the highest efficacy (73.2-89.6%), while the efficacy of vinclozolin was statistically significantly lower (48.7-63.4%) at both localities. However, qualitative and quantitative test of susceptibility to vinclozolin showed that all the isolates were susceptible to vinclozolin and that the reason for unsatisfactory efficacy should be primarily sought in inadequate fungicide application.

The Genus Botrytis and Botrytiscinerea species: Pathogenic, morphological and epidemiological characteristics

Species of the genus Botrytis occur wherever their hosts are grown, ranging from cold areas of Alaska to warm and dry areas in Israel. They have a necrotrophic life style which is often associated with phenology of the host plant. The genus comprises 22 species, most of which have a narrow host range. Polifagous species Botritys cinerea, a causal agent of grey mould disease, is the most important and the most extensively studied representative of this genus. More than 350 papers related to all aspects of the research of this necrotrophic pathogen are published each year. In this paper up-to-date knowledge about pathogenic, morphological and epidemic characteristics of the genus Botrytis and, particularly, species B. cinerea are summarized. Symptoms caused by B. cinerea on various plant species and various plant parts are shown. Morphological and genetic variability of the species is described. The possible mechanisms of variability, as well as the attempts to divide the species into Group I (B. „pseudocinerea“) and Group II (B. cinerea „sensu-stricto“) are pointed out.