Natural occurrence of fungal endophytes in cultivated cucumber plants in Syria, with emphasis on the entomopathogen Beauveria bassiana.

A survey was conducted to investigate endophytic Beauveria spp. and associated fungi in the tissues of cucumber plants (Cucumis sativus L.) cultivated in open fields and greenhouses in some regions of Syria during 2018-2019. Cultures of fungal endophytes belonging to nine genera were obtained (frequency %): Aspergillus (87.1%), Penicillium (41.23%), Fusarium (38.15%), Beauveria (12.83%), Trichoderma (9.87%), Colletotrichum (4.36%), Cladosporium (3.54%), Alternaria (2.79%), and Chaetomium (2.2%). Only Beauveria and Trichoderma cultures were entomopathogenic. Beauveria bassiana isolates were identified morphologically and molecularly from the stems, leaves, petioles, and fruits of cucumber plants collected at seven agricultural sites. Their ability to artificially colonize all cucumber plant parts was confirmed in the laboratory using soil drench. In this study, the natural association between the endophytic entomopathogenic fungus B. bassiana and cucumber plants is newly reported. In addition, a checklist of plant species reported in the literature to harbor this fungus is also provided.

First report of Stagonosporopsis pogostemonis causing leaf spot on cauliflower in Italy.

First report of Stagonosporopsis pogostemonis causing leaf spot on cauliflower in Italy Wassim Habib1, Mariangela Carlucci2, Roberto Fasano3, Franco Nigro1,2 † 1 Centre of Research, Experimentation and Training in Agriculture (CRSFA) - Basile Caramia, via Cisternino 281, Locorotondo, 70010 Bari, Italy 2 Department of Soil, Plant and Food Sciences, University of Bari - Aldo Moro, via Amendola 165A, 70126 Bari, Italy 3 Bioricerche, Phytopathological Lab, via Pio XI 30, Bellizzi, 84092 Salerno, Italy † Corresponding author: F. Nigro. Email: franco.nigro@uniba.it Keywords: Phoma-like, Multi-locus gene sequencing, Brassica oleracea L. var. botrytis, Campania. In September 2022, a severe foliar disease was observed on 2-3-week-old 'Corsaro' and 'Parthenon' seedlings of cauliflower (Brassica oleracea L. var. botrytis) grown under greenhouses in a nursery in Caserta province, Campania (Italy), with an incidence of 90% on about 150,000 plants. Affected plants showed numerous small, irregular, and depressed lesions bordered by a chlorotic halo on leaves that become necrotic, and later drop out giving a shot-hole effect (Fig. 1). Isolations were performed on leaf portions from 30 symptomatic samples which were surface-sterilised by dipping in 70 % ethanol for 30s, then in a sodium hypochlorite solution (1%) for 30s, and rinsed two times in distilled sterilised water. Tissue fragments were left to dry on sterile filter paper and then seeded on potato dextrose agar (PDA) amended with 0.5 g/L of streptomycin sulphate (Sigma-Aldrich S.R.L, Steinheim, Germany). After 6 to 8 days of incubation at 24 ± 1°C, colonies revealed a consistent growth of a Phoma-like fungus resembling Stagonosporopsis genus. On PDA, one-week-old colonies were dark olivaceous green to brown with white regular margin (Fig. 2B). Pycnidia (32-160 µm × 30-95 µm) were subglobose, and conidia (2.7-4.8 µm × 1-2.7 µm) ellipsoidal, with rounded ends, aseptate, with two polar guttules (Fig. 2C). All isolates had similar morphological features, therefore further analyses were performed on one representative strain (CRSFA.753.22). The multilocus phylogenetic approach using Maximum likelihood method and Tamura-Nei model on rDNA-ITS, TUB and RPB2 sequences (accession numbers: OQ318550, OQ326503, OQ326504) including reference strains of Stagonosporopsis (Dong et al. 2021) demonstrated that CRSFA.753.22 clustered with Stagonosporopsis pogostemonis Luo, Huang & Manawas holotype (ZHKUCC 21-0001) with 91% bootstrap support. ITS and RPB2 sequences had 100% BLAST match to ZHKUCC 21-0001, whereas TUB sequence had 99.69%. Pathogenicity tests were performed on 20 healthy 'Corsaro' plantlets. Leaves were surface sterilized with 70% alcohol and wounded with a sterile tip of a needle. A suspension of conidia and mycelial fragments, obtained from a 10-days-old colony of the strain CRSFA.753.22, was sprayed on the leaf surfaces at a distance of 20 cm. Five controls plants were inoculated with sterile distilled water. Plants were then covered with plastic bags and kept on the shelf in a growth chamber at 25° C, with a 12-h photoperiod. The first symptoms developed on leaves five days post-inoculation as irregular small brown spots, that were gradually expanded. Leaf tissues showed chlorosis which evolved into necrosis (Fig. 2A). Stagonosporopsis pogostemonis was consistently re-isolated from all diseased leaves, but not from control plants, thus fulfilling Koch's postulates. In Italy, Stagonosporopsis species have been reported as severe plant pathogens (Garibaldi et al. 2022; Guarnaccia et al. 2022), and this is the first report of the species S. pogostemonis in the country. References Dong, Z.Y., et al. 2021. Pathogens 10:1093. doi: 10.3390/pathogens10091093 Garibaldi, A., et al. 2022. Plant Pathol. J. 104:1157. doi: 10.1007/s42161-022-01138-7 Guarnaccia, V., et al. 2022. Plant Pathol. J. 104:1491. doi: 10.1007/s42161-022-01197-w.

First report of Ceratocystis ficicola causing wilt disease on common fig (Ficus carica) in Italy.

Common fig (Ficus carica L.) in is one of the most important crops in the Mediterranean area. In Italy, it is grown on a total area of 2118 ha. In Apulia (South-eastern Italy), the annual production of fig exceeds 3200 tons annually and together with olive and grapevine, they characterize the Apulian agricultural panorama. In September 2021, symptoms of a vascular wilt disease and, in severe cases, decline tree mortality were observed in Salento area (Apulia). Symptomatic Affected plants showed symptoms of leaf wilt and different stages of disease expression, which begins with leaf chlorosis on shoots, followed by wilting, extensive defoliation and twig dieback. On the main branches andlower part of the trunk and in some cases on lateral branches, bark cracks and cankers were observed and extended wood discoloration was detected in cross sections. In two orchards located in Salice Salentino (Site 1) and Squinzano (Site 2), where disease incidence exceeded 80%, 3-5 wood discs per tree were gathered from affected tissues from two (Site 1) and four (Site 2) trees. Isolations were performed on malt extract agar 2% amended with 0.5 g L-1 streptomycin sulfate. A Ceratocystis species was recovered from all samples trees with high frequencies (83.3%). Two-week-old colonies on potato dextrose agar showed black ascomata with 300-600 µm wide bases and 1100-2250 µm long necks. On the tips of the necks, ascospores (5-6x4-5 µm) exuded in creamy white sticky masses. Endoconidia (5-9x4.5-7 µm) were abundant, cylindrical, aseptate, and produced in chains. Two monoconidial representative strains CRSFA.Cer.033 (Site 1) and CRSFA.Cer.035 (Site 2) were deposited in the DISSPA Di.S.S.P.A. collection of the University of Bari. Species identification was done through sequence analyses of rDNA internal transcribed spacer region (ITS) using ITS5/ITS4 primers (White et al. 1990), elongation factor 1 alpha gene (TEF) using EF1/EF2 (O'Donnell et al. 1998) and RNA polymerase II gene (RPB2) gene using RPB2-5F/FRPB2fRPB2-7cR (Liu et al. 1999). Sequences were deposited in NCBI GenBank (accession numbers: OQ329983-OQ335969 (ITS), OQ352265-OQ352266 (TEF), OQ352268-OQ352267 (RPB2)). The sequences of both Apulian isolates were identical. BLAST searches revealed high similarity to the sequences of two isolates of Ceratocystis ficicola Kajitani and Masuya from Japan: ex-type CMW38543 and CMW38544, specifically 98.41% identity matching with KY685076 (ITS), 100% with KY685079 (TEF), and 99.87% with KY685083 and KY685082 (RPB2). Pathogenicity tests were conducted, on six common fig 'Dottato' seedlings by inoculating one year-old twigs with mycelium plugs (Bolboli et al. 2022). Control plants were inoculated with PDA plugs without mycelium. After one month, all inoculated twigs showed symptoms of wilt. Forty days post-inoculation, the bark of inoculated twigs was removed, and longitudinal and transverse sections revealed wood discolorations extending above and below the inoculation point. Ceratocystis ficicola was consistently reisolated from symptomatic tissues and identified as described above, thus fulfilling Koch's postulates. Neither symptoms nor positive isolations were observed in control seedlings. The pathogen was first described in 2011 in Japan (Kajitani and Masuya, 2011) where it currently affects all fig-producing areas. In 2018, it was reported for the first time in the EPPO region in Greece (Tsopelas et al., 2021), and to our knowledge, this is the first report on its detection in Italy. Since February 2022, C. ficicola has been included in the EPPO alert list because of its potential to cause tree mortality and the difficulty of its eradication. Regional national surveys are therefore urgently needed to determine its distribution in the fig growing areas of Italy and limit its spread. References Bolboli, Z., et al. 2022. Mycol. Prog. 21:89. doi: 10.1007/s11557-022-01834-9 Kajitani, Y., and Masuya, H., 2011. Mycoscience 52:349. doi: 10.1007/s10267-011-0116-5 Liu, Y. J., et al. 1999. Mol. Biol. and Evol. 16:1799. doi: 10.1093/oxfordjournals.molbev.a026092 O'Donnell, K., et al. 1998. Proc Natl Acad Sci USA 95:2044. doi: 10.1073/pnas.95.5.2044 Tsopelas, P., et al. 2021. Phytopathol. Mediterr. 60:337. doi: 10.36253/phyto-12794 White, T. J., et al. 1990. Page 315 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego. doi: 10.1016/0307-4412(91)90165-5.

Genetic Diversity of Verticillium dahliae Populations From Olive and Potato in Lebanon.

Verticillium dahliae is widely distributed in potato and olive fields in Lebanon, causing serious economic losses. However, little is known about the inoculum source, population structure, and genetic diversity of the pathogen or the mechanisms of dissemination within Lebanon. To understand the population structure, a total of 203 isolates sampled from olive (n = 78) and potato (n = 125) were characterized for species, mating type, and race, and the genetic relationships were delineated using 13 microsatellite markers. All isolates except one from potato were V. dahliae, with 55.1 and 12.1% race 1, and 43.6 and 83.1% race 2 in olive and potato, respectively. The genetic structure of the studied population was best described by two large and two small clusters. Membership in the two large clusters was determined by the presence or absence of the effector gene Ave1. Furthermore, genetic structure was moderately associated with the host of origin but was weakly associated with the geographic origin. All but four isolates represented by three multilocus haploid genotypes were MAT1-2. This study identified a clear lack of gene flow between virulence genotypes of V. dahliae despite the proximity of these cropping systems and the wide distribution of genetic diversity among hosts and geographic regions in Lebanon.

Long-Distance Spread of Verticillium dahliae Through Rivers and Irrigation Systems.

Verticillium dahliae Kleb. is a soilborne pathogen causing Verticillium wilt disease on several hosts. The pathogen survival structure (i.e., microsclerotia) can be efficiently spread by different dispersal methods. In the present study, the medium to long dispersal spread of the pathogen through rivers and irrigation canals was investigated. Samples of sediments (n = 29) were gathered from eight Lebanese rivers and three regional irrigation canals, in addition to samples of soil particles and plant residues (n = 14) from irrigation filters in commercial orchards. Specific conventional and real-time nested polymerase chain reaction assays detected the pathogen in six rivers-Al Kabir, Al Bared, Litani, Al Awali, Ostwan, and Litani South-and in all sampled canals-Ostwan, Al Bared, and Litani Canal 900. Starting DNA quantities ranged from 0.2 pg to 21.318 ng and an inoculum density, determined by a traditional plating method, varied between nondetectable and 0.2 microsclerotia/g. Viable V. dahliae microsclerotia were also found in residues collected from mesh-type irrigation filters of five commercial orchards. This study confirms that water is an important inoculum source of V. dahliae, being involved in the efficient spread of microsclerotia in Lebanese agricultural areas.

Diversity of Fusarium Species Isolated from Symptomatic Plants Belonging to a Wide Range of Agri-Food and Ornamental Crops in Lebanon.

Lebanon is a small Mediterranean country with different pedoclimatic conditions that allow the growth of both temperate and tropical plants. Currently, few studies are available on the occurrence and diversity of Fusarium species on Lebanese crops. A wide population of Fusarium strains was isolated from different symptomatic plants in the last 10 years. In the present investigation, a set of 134 representative strains were molecularly identified by sequencing the translation elongation factor, used in Fusarium as a barcoding gene. Great variability was observed, since the strains were grouped into nine different Fusarium Species Complexes (SCs). Fusarium oxysporum SC and Fusarium solani SC were the most frequent (53% and 24%, respectively). Members of important mycotoxigenic SCs were also detected: F. fujikuroi SC (7%), F. sambucinum SC (5%), F. incarnatum-equiseti SC (3%), and F. tricinctum SC (4%). Two strains belonging to F. lateritium SC, a single strain belonging to F. burgessii SC, and a single strain belonging to F. redolens SC were also detected. This paper reports, for the first time, the occurrence of several Fusarium species on Lebanese host plants. The clear picture of the Fusarium species distribution provided in this study can pose a basis for both a better understanding of the potential phytopathological and toxicological risks and planning future Fusarium management strategies in Lebanon.

Mycotoxin Profile and Phylogeny of Pathogenic Alternaria Species Isolated from Symptomatic Tomato Plants in Lebanon.

The tomato is one of the most consumed agri-food products in Lebanon. Several fungal pathogens, including Alternaria species, can infect tomato plants during the whole growing cycle. Alternaria infections cause severe production and economic losses in field and during storage. In addition, Alternaria species represent a serious toxicological risk since they are able to produce a wide range of mycotoxins, associated with different toxic activities on human and animal health. Several Alternaria species were detected on tomatoes, among which the most important are A. solani, A. alternata, and A. arborescens. A set of 49 Alternaria strains isolated from leaves and stems of diseased tomato plants were characterised by using a polyphasic approach. All strains were included in the recently defined phylogenetic Alternaria section and grouped in three well-separated sub-clades, namely A. alternata (24 out of 49), A. arborescens (12 out of 49), and A. mali morpho-species (12 out of 49). One strain showed high genetic similarity with an A.limoniasperae reference strain. Chemical analyses showed that most of the Alternaria strains, cultured on rice, were able to produce alternariol (AOH), alternariol methyl ether (AME), altenuene (ALT) and tenuazonic acid (TA), with values up to 5634, 16,006, 5156, and 4507 mg kg-1, respectively. In addition, 66% of the strains were able to co-produce simultaneously the four mycotoxins investigated. The pathogenicity test carried out on 10 Alternaria strains, representative of phylogenetic sub-clades, revealed that they were all pathogenic on tomato fruits. No significant difference among strains was observed, although A. alternata and A. arborescens strains were slightly more aggressive than A. mali morpho-species strains. This paper reports new insights on mycotoxin profiles, genetic variability, and pathogenicity of Alternaria species on tomatoes.

Occurrence and Characterization of Penicillium Species Isolated from Post-Harvest Apples in Lebanon.

The apple is one of the most important fruit tree crops in the Mediterranean region. Lebanon, in particular, is among the top apple producer countries in the Middle East; however, recently, several types of damage, particularly rot symptoms, have been detected on fruits in cold storage. This study aims to identify the causal agents of apple decay in Lebanese post-harvest facilities and characterize a set of 39 representative strains of the toxigenic fungus Penicillium. The results demonstrated that blue mould was the most frequent fungal disease associated with apples showing symptoms of decay after 3-4 months of storage at 0 °C, with an average frequency of 76.5% and 80.6% on cv. Red and cv. Golden Delicious apples, respectively. The morphological identification and phylogenetic analysis of benA gene showed that most Penicillium strains (87.2%) belong to P. expansum species whereas the remaining strains (12.8%) belong to P. solitum. Furthermore, 67.7% of P. expansum strains produced patulin when grown on apple puree for 14 days at 25 °C with values ranging from 10.7 mg kg-1 to 125.9 mg kg-1, whereas all P. solitum did not produce the mycotoxin. This study highlights the presence of Penicillium spp. and their related mycotoxin risk during apple storage and calls for the implementation of proper measures to decrease the risk of mycotoxin contamination of apple fruit products.