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Fungal diversity in tomato (Solanum lycopersicum) leaves and fruits in Russia

Fungal diversity in tomato (Solanum lycopersicum) leaves and fruits in Russia Short communication DOI: /10.5513/JCEA01/21.4.2869 Journal of Central European Agriculture, 2020, 21(4), p.809-816 1,2 3 1 1 1 Lyudmila Kokaeva , Elena Chudinova , Alexander Berezov , Maria Yarmeeva , Peotr Balabko , 1 1,3 Arseniy Belosokhov , Sergey Elansky (✉) Moscow Lomonosov State University, Moscow, Leninskiye Gory, 1/12, Russia 119991 Russian Potato Research Center, Lorh St., 23B, Kraskovo, Moscow Region, Russia 145023 Peoples Friendship University of Russia (RUDN University), Miklukho-Maklaya St., 6, Moscow, Russia 117198 ✉ Corresponding author: snelansky@gmail.com Received: April 18, 2020; accepted: July 27, 2020 ABSTRACT Sequencing of cloned PCR-amplified species-specific rDNA fragments and isolation of axenic cultures from tomato fruits was carried out to study the mycobiota of tomato leaves and fruits in European part of Russia. DNA was extracted from the leaves, and library of ITS region fragments was constructed in E. coli by cloning of PCR products. This survey revealed fourteen species associated with disease-aect ff ed leaves: Septoria lycopersici, Fulvia fulva (=Cladosporium fulvum), Didymella glomerata (=Phoma glomerata), Cladosporium herbarum, Podosphaera fusca, Neocamarosporium goegapense (=Phoma betae), Rhizoctonia solani, Candida albicans, Dioszegia hungarica, Cladosporium cladosporioides, Didymella lycopersici, Alternaria infectoria, Alternaria alternata, Cryptococcus tephrensis. In the leaves from healthy plants without any visible symptoms DNA of three species was found: Aspergillus versicolor, Alternaria alternata, Aureobasidium pullulans. Analysis of axenic cultures isolated from green diseased tomato fruits revealed fungal species: Alternaria alternata, Alternaria solani, Phomopsis phaseoli, Fusarium equiseti, Chaetomium cochliodes, Clonostachys sp., Irpex lacteus, Colletotrichum coccodes. This research provides new information on the mycobiota of tomato in Southern Russia, the main tomato producing region of the country. Keywords: mycobiota of tomato leaves, phylloplane, plant pathogenic fungi, fungal species identification, tomato diseases, fungal diversity INTRODUCTION about tomato leaves' mycobiota by now. Data from a Climatic changes and the intensive exchange of seed metagenomic analysis shows that mycobiota of tomato material lead to the emergence of new types of fungi phylloplane can be highly diverse. Fungal phyla identified associated with tomato plants. Foliar diseases cause from the surfaces of tomato leaves from Virginia, USA significant crop losses annually worldwide. Leaves can using 18S rRNA gene sequencing included the following also provide niches for the excitants of fruit infections. large taxonomic groups: Ascomycota, Basidiomycota, For example, Alternaria solani, Alternaria alternata, Chytridimycota, Glomeromycota, Zygomycota. Dominant Phytophthora infestans, Colletotrichum coccodes can cause fungal genera identified on aerial surfaces were Hypocrea, leaf spots, stem canker and fruit rot. Among fungi associated Aureobasidium, and Cryptococcus (Oesen tt et al., 2013). A with tomato plants, there are not only phytopathogens, high variety of fungi associated with tomato leaves was but also saprotrophs and mycotrophs, which can protect also noted in the work of Japanese researchers (Toju et the tomato from diseases and phytophages. On the other al., 2019). Another method used for mycobiota analysis hand, pathogenic or allergenic to human’s microorganisms involves the isolation and analysis of pure fungal cultures. can be in phylloplane too. Despite this, little is known Alternaria alternata, A. solani, Phytophthora infestans, 809 Short communication DOI: /10.5513/JCEA01/21.4.2869 Kokaeva et al.: Fungal diversity in tomato (Solanum lycopersicum) leaves and fruits in Russia... Septoria lycopercisi were isolated from tomato leaves in In the laboratory leaf surfaces were washed with sterile Pakistan (Chohan et al., 2016). In the work of Monaco water and dried. One simple leaf with small necrotic spots et al. (2001), it was shown that leaf position in the from every tested field was taken for analysis. DNA was canopy influenced the fungal population structure. Some extracted from the entire leaf using the conventional species, such as Epicoccum nigrum, Chaetomium globosum, hexadecyl-trimethyl-ammonium bromide (CTAB) Aspergillus sp., Trichoderma harzianum, T. polysporum and protocol with chloroform deproteinisation (Kokaeva et Penicillium spp., were more abundant on lower leaves, al., 2018a). DNA purity and concentration were measured while A. alternata, Cryptococcus luteolus, Rhodotorula sp., using a NanoDrop™ spectrophotometer. The fungal- Pleospora herbarum, Fusarium semitectum, F. oxysporum specific primer pair ITS1F and ITS4 (Gardes and Bruns, and Cladosporium cladosporioides were present in greater 1993; White et al., 1990) was used for the initial PCR numbers on leaves located in the upper and medium levels from leaf material. Aer ft amplification, the whole PCR (Monaco et al., 2001). However, both above methods product was analyzed in a 1.5% agarose gel stained with have their drawbacks. NGS sequencing rarely allows ethidium bromide. Amplification products were removed identification of species of microorganisms. Isolation of from the agarose gel with a sterile scalpel and cleaned axenic cultures does not make it possible to completely with Cleanup Standard kit (Evrogen, Russian Federation). study the species composition, since not all fungi can Cloning of PCR products was carried out using a pAL2-T grow on nutrient media. In this work the generation of Vector System kit (Evrogen, Russian Federation) and dh5α clone libraries of full-length internal transcribed spacer competent cells. Sterile water without addition of fungal region ITS1-5,8S-ITS2 was chosen as a powerful, culture- material served as a negative control. independent tool for observing fungal communities. This Ligations were performed by mixing the PCR product approach allows to identify fungal DNA up to the species with rapid ligation buffer, pAL2-T Vector, T4 DNA, and level. The combined use of this method with the isolation ligase. Bacterial transformations were performed by and analysis of axenic fungal cultures made it possible to adding the ligation product to dh5α competent cells maximally reveal the mycobiota associated with tomato and spreading on LB-agar plates, each with addition leaves and fruits. of ampicillin, X-Gal (5-bromo-4-chloro-3-indolyl-β-D- galactopyranoside) (Sigma), and IPTG (isopropyl β-D-1- MATERIALS AND METHODS thiogalactopyranoside) (Promega). Plates were incubated Analysis of leaves at 37 ºC for 24 h. Bacterial cells carrying the vector with Leaves with spots and necrosis were collected in July- successfully ligated PCR product form white colonies August, 2014 by the authors themselves from field-grown under the conditions described above. plants in five regions of European Russia (Figure 1, Table DNA was extracted from white colonies and 1). In total, samples from 4 commercial fields and 3 small ITS regions were amplified using M13 primers private gardens were investigated. In Rostov region and (M13f-TGTAAAACGACGGCCAGT/ M13r - Krasnodar territory samples were collected in commercial CAGGAAACAGCTATGAC). PCR products were analyzed fields; in Voronezh region and Stavropol territory – from in 1.5% agarose gel to verify the presence of amplification small kitchen-gardens. Leaves with no visible symptoms products of the correct size. were collected from healthy tomato plants in small At least 40 clones from each leaf sample were tested private garden in the national park “Kurshskaya kosa” in with digestion using MspI restriction enzyme (Sibenzim, Kaliningrad region. Leaves were collected from the top Russia). RFLP products were analyzed in 2% agarose third of each selected plant. Fresh leaves were fixed in gel. Tested clones had different rDNA PCR restriction 70% alcohol immediately aer ft sampling. For analysis one paerns (F tt igure 2). simple leaf from one field or garden was taken. 810 Short communication DOI: /10.5513/JCEA01/21.4.2869 Kokaeva et al.: Fungal diversity in tomato (Solanum lycopersicum) leaves and fruits in Russia... Figure 1. Collection sites (see Table 1) For sequencing 20-30 clones with insertions (including Analysis of fruits clones with different restriction paerns tt ) from every leaf In the field in Krasnodar Territory (site 5, Figure 1, sample were used. A total of 200 clones were sequenced. Table 1), mycobiota of tomato fruits was analyzed by the Sequencing reactions were performed following the method of isolation and analysis of axenic cultures. Green BigDye terminator protocol (ABI Prism) with M13 fruits with dark dry spots on the surface were used for primers. Sequences were analyzed by the Blast program tests. Fruits were washed carefully, and surface sterilized at the Genbank (NCBI, USA) and at UNITE database. with 70% alcohol. Fruits slices were placed in moist Both programs yielded identical results for almost all chambers. Mycelium or spores were taken from alive tested clones. 811 Short communication DOI: /10.5513/JCEA01/21.4.2869 Kokaeva et al.: Fungal diversity in tomato (Solanum lycopersicum) leaves and fruits in Russia... The most commonly occurring species were A. alternata (30% of all clones), S. lycopersici (9%), F. fulva (8%). N. goegapense, R. solani, C. albicans, D. hungarica, C. cladosporioides, D. lycopersici, A. infectoria, C. tephrensis were recovered with low frequencies. In the leaves from healthy plants without any visible symptoms DNA of three species was found: Aspergillus versicolor (Vuill.) Tirab., Annali, A. alternata, Aureobasidium pullulans (de Bary & Löwenthal) G. Arnaud. The number of species per one sample (simple leaf) Lines1, 3, 5, 6: Septoria lycopersici; 2: Podosphaera fusca; 4: Alternaria varied from two to six. The largest number of species alternata; M – 1 kb DNA Ladder was found in samples from Rostov region (sites 3 and Figure 2. Restriction paerns tt of 700-bp fragment amplified by 6, Table 1), four species were found in one sample from M13 primers and digested with MspI Kislovodsk (site 7), other samples contain DNA of 2-3 tissue with a needle under the microscope and placed on species (Table 1). Similar results were obtained aer ft a Petri dish with wort agar with the addition of penicillin. analysis of potato (Solanum tuberosum) and bitter-sweet In total, 45 fruits were investigated. Isolation of fungal nightshade (Solanum dulcamara) leaves (Kokaeva et al., strains, DNA extraction, PCR amplification, extraction of 2018b, Kokaeva et al., 2019). amplicons, and sequencing were performed as described in Kutuzova et al. (2017). The species affiliation of tested Analysis of tomato fruits mycobiota isolates was determined by cultural and morphological Several fungal species were identified aer ft the characters and by sequences of species-specific DNA analysis of axenic cultures isolated from green diseased region ITS1-5,8S-ITS2 (primers ITS5 and ITS4, or ITS1F tomato fruits. A. alternata (GenBank accession number and ITS4) (White et al., 1990; Gardes and Bruns, 1993). KU245685) was detected in most of the studied fruits. RESULTS Other identified fungal species were Phomopsis phaseoli (Desm.) Sacc. (MH412692), Alternaria solani Sorauer Analysis of fungal diversity in tomato leaves using (KY496637), Fusarium equiseti (Corda) Sacc. (MT588081), sequencing of cloned PCR-amplified rDNA Chaetomium cochliodes Palliser (MT279444), Clonostachys In this study, a total of 14 unique rDNA sequences sp. (MT588112), Irpex lacteus (Fr.) Fr. (MT276332), 482-682 bp long were obtained. Aer ft sequence analysis Colletotrichum coccodes (Wallr.) S. Hughes (MT292616). specific DNA regions of fourteen species were revealed in blighted leaves: Septoria lycopersici Speg., Fulvia DISCUSSION fulva (Cooke) Cif. (=Cladosporium fulvum), Didymella South regions, including Krasnodar and Stavropol glomerata (Corda) Qian Chen & L. Cai (=Phoma glomerata), territories, Rostov region, are the main tomato producing Cladosporium herbarum (Pers.) Link, Podosphaera fusca areas in Russia. Development of fungal diseases cause a (Fr.) U. Braun & Shishko, ff Neocamarosporium goegapense great crop loss. Appearance of new fungal species leads to Crous & M.J. Wingf. (=Phoma betae), Rhizoctonia solani J.G. decrease of efficiency of traditional protection schemes. Kühn, Candida albicans (C.P. Robin) Berkhout, Dioszegia Novel species of fungal pathogens can be resistant to hungarica Zsolt, Cladosporium cladosporioides (Fresen.) traditionally applied plant protection products. G.A. de Vries, Didymella lycopersici Kleb., Alternaria infectoria E.G. Simmons, Alternaria alternata (Fr.) Keissl., In this research both widespread on tomato and Cryptococcus tephrensis Vishniac. rare, not previously noted in Russia, fungal species were 812 Short communication DOI: /10.5513/JCEA01/21.4.2869 Kokaeva et al.: Fungal diversity in tomato (Solanum lycopersicum) leaves and fruits in Russia... Table 1. Identified fungal species in leaf samples using sequencing of cloned PCR-amplified rDNA Indication on a Identified species, GenBank accession Collection site Description of plants on the site map (Figure 1) numbers 1 Kaliningrad region. Small private garden. Plants green. Leaves Aspergillus versicolor MT279494 Kurshskaya kosa national without any disease symptoms. park. Control plot with Alternaria alternata KU245685 healthy plants. Aureobasidium pullulans MT279495 2 Voronezh region. Private garden. Plants were green and Cladosporium herbarum KU245683 Paninsky district. weakly aect ff ed. Leaves were green with dry necrotic lesions. Rhizoctonia solani KU245690 Alternaria alternata (KU245685) 3 Rostov region, near Commercial field. Strong aection o ff f Dioszegia hungarica KU245691 Rostov-na-Donu city. individual plants. Many ripen fruits had rot infection. Leaves were green with dry Cladosporium herbarum (KU245683) necroses. Sprinkler irrigation. Neocamarosporium goegapense KU245684 Alternaria alternata (KU245685) Rhizoctonia solani (KU245690) Candida albicans KU245692 4 The Krasnodar territory, Commercial field. Plants were green and Fulvia fulva KU245688 Temryuk district. weakly aect ff ed. Leaves were green with rare dry necrotic lesions. Drip irrigation. Didymella lycopersici MT279496 5 The Krasnodar territory, Commercial field. Strong aection o ff f Fulvia fulva (KU245688) Slavyansk-na-Kubani most plants and fruits. Leaves with many district. necrotic lesions. Sprinkler irrigation. Peyronellaea glomerata KU245689 Alternaria alternata KU245685 6 Rostov region, Armavir Commercial field. Plants were weakly Alternaria alternata (KU245685) district aect ff ed. Ripen fruits without any rots. Green leaves with dry necroses were Septoria lycopersici KU245686 collected. Sprinkler irrigation. Cladosporium herbarum (KU245683) Alternaria infectoria MK131038 Cryptococcus tephrensis KU245693 7 Stavropol territory, Private garden 1. Plants were mainly green Septoria lycopersici (KU245686) Kislovodsk city. and weakly aect ff ed. Leaves were green with dry necrotic lesions. Podosphaera fusca KU245687 Alternaria alternata (KU245685) Cladosporium cladosporioides MK131039 7 Stavropol territory, Private garden 2. Plants were mainly green Septoria lycopersici (KU245686) Kislovodsk city. and weakly aect ff ed. Leaves were green with dry necrotic lesions. Podosphaera fusca (KU245687) Alternaria alternata (KU245685) sequence of the sample completely (100%) identical to submitted number in parenthesis 813 Short communication DOI: /10.5513/JCEA01/21.4.2869 Kokaeva et al.: Fungal diversity in tomato (Solanum lycopersicum) leaves and fruits in Russia... detected. S. lycopersici, P. fulva, D. lycopersici, A. infectoria, 2018; Manning, 1980). P. phaseoli is known as a soybean A. alternata — these ubiquitous pathogenic species pathogen; it was first discovered on tomato (Elansky et al., occur on tomato all over the world. The pathogenicity 2020). F. equiseti was found as a tomato pathogen in Asian of A. alternata in relation to different tomato varieties countries (Akbar et al., 2018; Kamlesh and Ramchandra, was studied in an earlier research (Kudryavtseva et al., 2017). C. cochliodes and Clonostachys sp. — saprotrophic 2017). Some identified species (P. fusca, N. goegapense, P. soil fungi entering antagonistic relations with many soil glomerata) are not typical for phytopathogenic mycobiota microorganisms. The biocidal activity of Clonostachys of tomato and were never observed in Russia. P. fusca against nematodes (Silva et al., 2015) and some fungal is one of the causative agents of powdery mildew in phytopathogens (Borges et al., 2015) was shown. Strains cucurbits and some other angiosperm plants (Pérez- of Chaetomium are also used as biofungicide (Soytong et García et al., 2009). This species was never previously al., 2001). I. lacteus — basidiomycete, a white rot fungus, reported on tomato plants. However, this pathogen was is not known as tomato pathogen. found on Solanaceae plants: on Nicotiana alata in USSR In this research it was evaluated the growth ability of (Braun, 1995), and on Solanum macrocarpum in Korea several strains on whole tomato fruits and its slices in a (Cho and Shin, 2004). N. goegapense (= Phoma betae) is a moist chamber. Symptomless, detached green tomato pathogen causing "zonal" leaf spot. The species was found fruits, surface sterilized with ethanol (70%) and their slices in tomato plants in India (Mathur, 1979). Peyronellaea were used for this test. According to our experiments, glomerata (= Phoma glomerata) have wide range of host Clonostachys sp., C. cochliodes, P. phaseoli, and I. lacteus plants. The pathogenicity of this microorganism was were not able to penetrate the tomato epidermis and proved on potato tubers and leaves (Kranz, 1963). P. infect fruits, but they developed well on fruits' cuttings. glomerata was detected on tomato in the Netherlands It seems that these fungi can parasitize on tomato fruits and potato in Germany (Aveskamp et al., 2010). This when a crack occurs on their surface. F. equiseti showed paper is, to available knowledge, the first report of P. great aggressiveness, it can infect the tomato fruits glomerata and N. goegapense on tomato in Russia. R. solani through the epidermis (Chudinova et al., 2020). is noted as the causative agent of foliar blight of tomato A. solani and C. coccodes were not identified in cloning (Ivors et al., 2009), and it is noted that lesions are similar test, but present on the diseased fruits. In previous to the lesions of A. alternata. R. solani can inoculate stems research with Alternaria spp. PCR identification (Kokaeva and roots of Solanum lycopersicum (Pourmahdi and Taheri, et al., 2018a) was found that 40% of leaf samples contain 2015; Manning, 1980). A. solani DNA in Temryuk (location 4, Figure1), 35% — in On a healthy leaf from control plot in Kaliningrad Kislovodsk (7), 7% in Rostov (6), 0% in Armavir (6). Our region (site 1) A. pullulans, a yeast-like saprophytic fungus, research with PCR identification of C. coccodes (Belov was found. It naturally inhabits plant and fruit surfaces. et al., 2018) revealed 54% of leaf samples with DNA of It was tested as a potential biocontrol agent against a this fungus in Rostov (3), 28% in Slavyansk-na-Kubani wide range of pathogenic fungi. Italian researchers tried (5), 4% in Armavir (6), 0% in Temryuk (4). In present to use this fungus as a potential biocontrol agent against research it was analyzed only one leaf sample per field; Phytophthora infestans on tomato (Francesco et al., 2017). possibly, it was too small to find C. coccodes or A. solani. Investigation of tomato fruits revealed several Microscopic exploration of large amount of tomato fruits widespread (A. alternata, A. solani, C. coccodes) and new in site 5 revealed these two pathogens. It shows that field tomato pathogens (P. phaseoli, F. equiseti, I. lacteus) and research with sequencing of cloned PCR-amplified rDNA saprotrophic species C. cochliodes and Clonostachys sp. A. can be implemented for research of mycobiota and search alternata was detected in most of the studied fruits. C. for new fungal species on the plant, but not for screening coccodes is a widespread tomato pathogen (Belov et al., for dangerous pathogens. 814 Short communication DOI: /10.5513/JCEA01/21.4.2869 Kokaeva et al.: Fungal diversity in tomato (Solanum lycopersicum) leaves and fruits in Russia... Gardes, M., Bruns, T. D. (1993). ITS primers with enhanced specificity CONCLUSIONS for basidiomycetes-application to the identification of mycorrhizae and rusts. Molecular Ecology, 2 (2), 113-118. This research provides new information on the Ivors, K. L., Bartz, F. E., Toda, T., Naito, S., Cubeta, M. A. (2009) First mycobiota of tomato leaves. 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(2019) Leaf-associated microbiomes of graed t ft omato plants. Scientific Reports, 9, 1787. DOI: https://doi.org/10.1038/s41598-018-38344-2 Powered by TCPDF (www.tcpdf.org) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Central European Agriculture Unpaywall

Fungal diversity in tomato (Solanum lycopersicum) leaves and fruits in Russia

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Short communication DOI: /10.5513/JCEA01/21.4.2869 Journal of Central European Agriculture, 2020, 21(4), p.809-816 1,2 3 1 1 1 Lyudmila Kokaeva , Elena Chudinova , Alexander Berezov , Maria Yarmeeva , Peotr Balabko , 1 1,3 Arseniy Belosokhov , Sergey Elansky (✉) Moscow Lomonosov State University, Moscow, Leninskiye Gory, 1/12, Russia 119991 Russian Potato Research Center, Lorh St., 23B, Kraskovo, Moscow Region, Russia 145023 Peoples Friendship University of Russia (RUDN University), Miklukho-Maklaya St., 6, Moscow, Russia 117198 ✉ Corresponding author: snelansky@gmail.com Received: April 18, 2020; accepted: July 27, 2020 ABSTRACT Sequencing of cloned PCR-amplified species-specific rDNA fragments and isolation of axenic cultures from tomato fruits was carried out to study the mycobiota of tomato leaves and fruits in European part of Russia. DNA was extracted from the leaves, and library of ITS region fragments was constructed in E. coli by cloning of PCR products. This survey revealed fourteen species associated with disease-aect ff ed leaves: Septoria lycopersici, Fulvia fulva (=Cladosporium fulvum), Didymella glomerata (=Phoma glomerata), Cladosporium herbarum, Podosphaera fusca, Neocamarosporium goegapense (=Phoma betae), Rhizoctonia solani, Candida albicans, Dioszegia hungarica, Cladosporium cladosporioides, Didymella lycopersici, Alternaria infectoria, Alternaria alternata, Cryptococcus tephrensis. In the leaves from healthy plants without any visible symptoms DNA of three species was found: Aspergillus versicolor, Alternaria alternata, Aureobasidium pullulans. Analysis of axenic cultures isolated from green diseased tomato fruits revealed fungal species: Alternaria alternata, Alternaria solani, Phomopsis phaseoli, Fusarium equiseti, Chaetomium cochliodes, Clonostachys sp., Irpex lacteus, Colletotrichum coccodes. This research provides new information on the mycobiota of tomato in Southern Russia, the main tomato producing region of the country. Keywords: mycobiota of tomato leaves, phylloplane, plant pathogenic fungi, fungal species identification, tomato diseases, fungal diversity INTRODUCTION about tomato leaves' mycobiota by now. Data from a Climatic changes and the intensive exchange of seed metagenomic analysis shows that mycobiota of tomato material lead to the emergence of new types of fungi phylloplane can be highly diverse. Fungal phyla identified associated with tomato plants. Foliar diseases cause from the surfaces of tomato leaves from Virginia, USA significant crop losses annually worldwide. Leaves can using 18S rRNA gene sequencing included the following also provide niches for the excitants of fruit infections. large taxonomic groups: Ascomycota, Basidiomycota, For example, Alternaria solani, Alternaria alternata, Chytridimycota, Glomeromycota, Zygomycota. Dominant Phytophthora infestans, Colletotrichum coccodes can cause fungal genera identified on aerial surfaces were Hypocrea, leaf spots, stem canker and fruit rot. Among fungi associated Aureobasidium, and Cryptococcus (Oesen tt et al., 2013). A with tomato plants, there are not only phytopathogens, high variety of fungi associated with tomato leaves was but also saprotrophs and mycotrophs, which can protect also noted in the work of Japanese researchers (Toju et the tomato from diseases and phytophages. On the other al., 2019). Another method used for mycobiota analysis hand, pathogenic or allergenic to human’s microorganisms involves the isolation and analysis of pure fungal cultures. can be in phylloplane too. Despite this, little is known Alternaria alternata, A. solani, Phytophthora infestans, 809 Short communication DOI: /10.5513/JCEA01/21.4.2869 Kokaeva et al.: Fungal diversity in tomato (Solanum lycopersicum) leaves and fruits in Russia... Septoria lycopercisi were isolated from tomato leaves in In the laboratory leaf surfaces were washed with sterile Pakistan (Chohan et al., 2016). In the work of Monaco water and dried. One simple leaf with small necrotic spots et al. (2001), it was shown that leaf position in the from every tested field was taken for analysis. DNA was canopy influenced the fungal population structure. Some extracted from the entire leaf using the conventional species, such as Epicoccum nigrum, Chaetomium globosum, hexadecyl-trimethyl-ammonium bromide (CTAB) Aspergillus sp., Trichoderma harzianum, T. polysporum and protocol with chloroform deproteinisation (Kokaeva et Penicillium spp., were more abundant on lower leaves, al., 2018a). DNA purity and concentration were measured while A. alternata, Cryptococcus luteolus, Rhodotorula sp., using a NanoDrop™ spectrophotometer. The fungal- Pleospora herbarum, Fusarium semitectum, F. oxysporum specific primer pair ITS1F and ITS4 (Gardes and Bruns, and Cladosporium cladosporioides were present in greater 1993; White et al., 1990) was used for the initial PCR numbers on leaves located in the upper and medium levels from leaf material. Aer ft amplification, the whole PCR (Monaco et al., 2001). However, both above methods product was analyzed in a 1.5% agarose gel stained with have their drawbacks. NGS sequencing rarely allows ethidium bromide. Amplification products were removed identification of species of microorganisms. Isolation of from the agarose gel with a sterile scalpel and cleaned axenic cultures does not make it possible to completely with Cleanup Standard kit (Evrogen, Russian Federation). study the species composition, since not all fungi can Cloning of PCR products was carried out using a pAL2-T grow on nutrient media. In this work the generation of Vector System kit (Evrogen, Russian Federation) and dh5α clone libraries of full-length internal transcribed spacer competent cells. Sterile water without addition of fungal region ITS1-5,8S-ITS2 was chosen as a powerful, culture- material served as a negative control. independent tool for observing fungal communities. This Ligations were performed by mixing the PCR product approach allows to identify fungal DNA up to the species with rapid ligation buffer, pAL2-T Vector, T4 DNA, and level. The combined use of this method with the isolation ligase. Bacterial transformations were performed by and analysis of axenic fungal cultures made it possible to adding the ligation product to dh5α competent cells maximally reveal the mycobiota associated with tomato and spreading on LB-agar plates, each with addition leaves and fruits. of ampicillin, X-Gal (5-bromo-4-chloro-3-indolyl-β-D- galactopyranoside) (Sigma), and IPTG (isopropyl β-D-1- MATERIALS AND METHODS thiogalactopyranoside) (Promega). Plates were incubated Analysis of leaves at 37 ºC for 24 h. Bacterial cells carrying the vector with Leaves with spots and necrosis were collected in July- successfully ligated PCR product form white colonies August, 2014 by the authors themselves from field-grown under the conditions described above. plants in five regions of European Russia (Figure 1, Table DNA was extracted from white colonies and 1). In total, samples from 4 commercial fields and 3 small ITS regions were amplified using M13 primers private gardens were investigated. In Rostov region and (M13f-TGTAAAACGACGGCCAGT/ M13r - Krasnodar territory samples were collected in commercial CAGGAAACAGCTATGAC). PCR products were analyzed fields; in Voronezh region and Stavropol territory – from in 1.5% agarose gel to verify the presence of amplification small kitchen-gardens. Leaves with no visible symptoms products of the correct size. were collected from healthy tomato plants in small At least 40 clones from each leaf sample were tested private garden in the national park “Kurshskaya kosa” in with digestion using MspI restriction enzyme (Sibenzim, Kaliningrad region. Leaves were collected from the top Russia). RFLP products were analyzed in 2% agarose third of each selected plant. Fresh leaves were fixed in gel. Tested clones had different rDNA PCR restriction 70% alcohol immediately aer ft sampling. For analysis one paerns (F tt igure 2). simple leaf from one field or garden was taken. 810 Short communication DOI: /10.5513/JCEA01/21.4.2869 Kokaeva et al.: Fungal diversity in tomato (Solanum lycopersicum) leaves and fruits in Russia... Figure 1. Collection sites (see Table 1) For sequencing 20-30 clones with insertions (including Analysis of fruits clones with different restriction paerns tt ) from every leaf In the field in Krasnodar Territory (site 5, Figure 1, sample were used. A total of 200 clones were sequenced. Table 1), mycobiota of tomato fruits was analyzed by the Sequencing reactions were performed following the method of isolation and analysis of axenic cultures. Green BigDye terminator protocol (ABI Prism) with M13 fruits with dark dry spots on the surface were used for primers. Sequences were analyzed by the Blast program tests. Fruits were washed carefully, and surface sterilized at the Genbank (NCBI, USA) and at UNITE database. with 70% alcohol. Fruits slices were placed in moist Both programs yielded identical results for almost all chambers. Mycelium or spores were taken from alive tested clones. 811 Short communication DOI: /10.5513/JCEA01/21.4.2869 Kokaeva et al.: Fungal diversity in tomato (Solanum lycopersicum) leaves and fruits in Russia... The most commonly occurring species were A. alternata (30% of all clones), S. lycopersici (9%), F. fulva (8%). N. goegapense, R. solani, C. albicans, D. hungarica, C. cladosporioides, D. lycopersici, A. infectoria, C. tephrensis were recovered with low frequencies. In the leaves from healthy plants without any visible symptoms DNA of three species was found: Aspergillus versicolor (Vuill.) Tirab., Annali, A. alternata, Aureobasidium pullulans (de Bary & Löwenthal) G. Arnaud. The number of species per one sample (simple leaf) Lines1, 3, 5, 6: Septoria lycopersici; 2: Podosphaera fusca; 4: Alternaria varied from two to six. The largest number of species alternata; M – 1 kb DNA Ladder was found in samples from Rostov region (sites 3 and Figure 2. Restriction paerns tt of 700-bp fragment amplified by 6, Table 1), four species were found in one sample from M13 primers and digested with MspI Kislovodsk (site 7), other samples contain DNA of 2-3 tissue with a needle under the microscope and placed on species (Table 1). Similar results were obtained aer ft a Petri dish with wort agar with the addition of penicillin. analysis of potato (Solanum tuberosum) and bitter-sweet In total, 45 fruits were investigated. Isolation of fungal nightshade (Solanum dulcamara) leaves (Kokaeva et al., strains, DNA extraction, PCR amplification, extraction of 2018b, Kokaeva et al., 2019). amplicons, and sequencing were performed as described in Kutuzova et al. (2017). The species affiliation of tested Analysis of tomato fruits mycobiota isolates was determined by cultural and morphological Several fungal species were identified aer ft the characters and by sequences of species-specific DNA analysis of axenic cultures isolated from green diseased region ITS1-5,8S-ITS2 (primers ITS5 and ITS4, or ITS1F tomato fruits. A. alternata (GenBank accession number and ITS4) (White et al., 1990; Gardes and Bruns, 1993). KU245685) was detected in most of the studied fruits. RESULTS Other identified fungal species were Phomopsis phaseoli (Desm.) Sacc. (MH412692), Alternaria solani Sorauer Analysis of fungal diversity in tomato leaves using (KY496637), Fusarium equiseti (Corda) Sacc. (MT588081), sequencing of cloned PCR-amplified rDNA Chaetomium cochliodes Palliser (MT279444), Clonostachys In this study, a total of 14 unique rDNA sequences sp. (MT588112), Irpex lacteus (Fr.) Fr. (MT276332), 482-682 bp long were obtained. Aer ft sequence analysis Colletotrichum coccodes (Wallr.) S. Hughes (MT292616). specific DNA regions of fourteen species were revealed in blighted leaves: Septoria lycopersici Speg., Fulvia DISCUSSION fulva (Cooke) Cif. (=Cladosporium fulvum), Didymella South regions, including Krasnodar and Stavropol glomerata (Corda) Qian Chen & L. Cai (=Phoma glomerata), territories, Rostov region, are the main tomato producing Cladosporium herbarum (Pers.) Link, Podosphaera fusca areas in Russia. Development of fungal diseases cause a (Fr.) U. Braun & Shishko, ff Neocamarosporium goegapense great crop loss. Appearance of new fungal species leads to Crous & M.J. Wingf. (=Phoma betae), Rhizoctonia solani J.G. decrease of efficiency of traditional protection schemes. Kühn, Candida albicans (C.P. Robin) Berkhout, Dioszegia Novel species of fungal pathogens can be resistant to hungarica Zsolt, Cladosporium cladosporioides (Fresen.) traditionally applied plant protection products. G.A. de Vries, Didymella lycopersici Kleb., Alternaria infectoria E.G. Simmons, Alternaria alternata (Fr.) Keissl., In this research both widespread on tomato and Cryptococcus tephrensis Vishniac. rare, not previously noted in Russia, fungal species were 812 Short communication DOI: /10.5513/JCEA01/21.4.2869 Kokaeva et al.: Fungal diversity in tomato (Solanum lycopersicum) leaves and fruits in Russia... Table 1. Identified fungal species in leaf samples using sequencing of cloned PCR-amplified rDNA Indication on a Identified species, GenBank accession Collection site Description of plants on the site map (Figure 1) numbers 1 Kaliningrad region. Small private garden. Plants green. Leaves Aspergillus versicolor MT279494 Kurshskaya kosa national without any disease symptoms. park. Control plot with Alternaria alternata KU245685 healthy plants. Aureobasidium pullulans MT279495 2 Voronezh region. Private garden. Plants were green and Cladosporium herbarum KU245683 Paninsky district. weakly aect ff ed. Leaves were green with dry necrotic lesions. Rhizoctonia solani KU245690 Alternaria alternata (KU245685) 3 Rostov region, near Commercial field. Strong aection o ff f Dioszegia hungarica KU245691 Rostov-na-Donu city. individual plants. Many ripen fruits had rot infection. Leaves were green with dry Cladosporium herbarum (KU245683) necroses. Sprinkler irrigation. Neocamarosporium goegapense KU245684 Alternaria alternata (KU245685) Rhizoctonia solani (KU245690) Candida albicans KU245692 4 The Krasnodar territory, Commercial field. Plants were green and Fulvia fulva KU245688 Temryuk district. weakly aect ff ed. Leaves were green with rare dry necrotic lesions. Drip irrigation. Didymella lycopersici MT279496 5 The Krasnodar territory, Commercial field. Strong aection o ff f Fulvia fulva (KU245688) Slavyansk-na-Kubani most plants and fruits. Leaves with many district. necrotic lesions. Sprinkler irrigation. Peyronellaea glomerata KU245689 Alternaria alternata KU245685 6 Rostov region, Armavir Commercial field. Plants were weakly Alternaria alternata (KU245685) district aect ff ed. Ripen fruits without any rots. Green leaves with dry necroses were Septoria lycopersici KU245686 collected. Sprinkler irrigation. Cladosporium herbarum (KU245683) Alternaria infectoria MK131038 Cryptococcus tephrensis KU245693 7 Stavropol territory, Private garden 1. Plants were mainly green Septoria lycopersici (KU245686) Kislovodsk city. and weakly aect ff ed. Leaves were green with dry necrotic lesions. Podosphaera fusca KU245687 Alternaria alternata (KU245685) Cladosporium cladosporioides MK131039 7 Stavropol territory, Private garden 2. Plants were mainly green Septoria lycopersici (KU245686) Kislovodsk city. and weakly aect ff ed. Leaves were green with dry necrotic lesions. Podosphaera fusca (KU245687) Alternaria alternata (KU245685) sequence of the sample completely (100%) identical to submitted number in parenthesis 813 Short communication DOI: /10.5513/JCEA01/21.4.2869 Kokaeva et al.: Fungal diversity in tomato (Solanum lycopersicum) leaves and fruits in Russia... detected. S. lycopersici, P. fulva, D. lycopersici, A. infectoria, 2018; Manning, 1980). P. phaseoli is known as a soybean A. alternata — these ubiquitous pathogenic species pathogen; it was first discovered on tomato (Elansky et al., occur on tomato all over the world. The pathogenicity 2020). F. equiseti was found as a tomato pathogen in Asian of A. alternata in relation to different tomato varieties countries (Akbar et al., 2018; Kamlesh and Ramchandra, was studied in an earlier research (Kudryavtseva et al., 2017). C. cochliodes and Clonostachys sp. — saprotrophic 2017). Some identified species (P. fusca, N. goegapense, P. soil fungi entering antagonistic relations with many soil glomerata) are not typical for phytopathogenic mycobiota microorganisms. The biocidal activity of Clonostachys of tomato and were never observed in Russia. P. fusca against nematodes (Silva et al., 2015) and some fungal is one of the causative agents of powdery mildew in phytopathogens (Borges et al., 2015) was shown. Strains cucurbits and some other angiosperm plants (Pérez- of Chaetomium are also used as biofungicide (Soytong et García et al., 2009). This species was never previously al., 2001). I. lacteus — basidiomycete, a white rot fungus, reported on tomato plants. However, this pathogen was is not known as tomato pathogen. found on Solanaceae plants: on Nicotiana alata in USSR In this research it was evaluated the growth ability of (Braun, 1995), and on Solanum macrocarpum in Korea several strains on whole tomato fruits and its slices in a (Cho and Shin, 2004). N. goegapense (= Phoma betae) is a moist chamber. Symptomless, detached green tomato pathogen causing "zonal" leaf spot. The species was found fruits, surface sterilized with ethanol (70%) and their slices in tomato plants in India (Mathur, 1979). Peyronellaea were used for this test. According to our experiments, glomerata (= Phoma glomerata) have wide range of host Clonostachys sp., C. cochliodes, P. phaseoli, and I. lacteus plants. The pathogenicity of this microorganism was were not able to penetrate the tomato epidermis and proved on potato tubers and leaves (Kranz, 1963). P. infect fruits, but they developed well on fruits' cuttings. glomerata was detected on tomato in the Netherlands It seems that these fungi can parasitize on tomato fruits and potato in Germany (Aveskamp et al., 2010). This when a crack occurs on their surface. F. equiseti showed paper is, to available knowledge, the first report of P. great aggressiveness, it can infect the tomato fruits glomerata and N. goegapense on tomato in Russia. R. solani through the epidermis (Chudinova et al., 2020). is noted as the causative agent of foliar blight of tomato A. solani and C. coccodes were not identified in cloning (Ivors et al., 2009), and it is noted that lesions are similar test, but present on the diseased fruits. In previous to the lesions of A. alternata. R. solani can inoculate stems research with Alternaria spp. PCR identification (Kokaeva and roots of Solanum lycopersicum (Pourmahdi and Taheri, et al., 2018a) was found that 40% of leaf samples contain 2015; Manning, 1980). A. solani DNA in Temryuk (location 4, Figure1), 35% — in On a healthy leaf from control plot in Kaliningrad Kislovodsk (7), 7% in Rostov (6), 0% in Armavir (6). Our region (site 1) A. pullulans, a yeast-like saprophytic fungus, research with PCR identification of C. coccodes (Belov was found. It naturally inhabits plant and fruit surfaces. et al., 2018) revealed 54% of leaf samples with DNA of It was tested as a potential biocontrol agent against a this fungus in Rostov (3), 28% in Slavyansk-na-Kubani wide range of pathogenic fungi. Italian researchers tried (5), 4% in Armavir (6), 0% in Temryuk (4). In present to use this fungus as a potential biocontrol agent against research it was analyzed only one leaf sample per field; Phytophthora infestans on tomato (Francesco et al., 2017). possibly, it was too small to find C. coccodes or A. solani. Investigation of tomato fruits revealed several Microscopic exploration of large amount of tomato fruits widespread (A. alternata, A. solani, C. coccodes) and new in site 5 revealed these two pathogens. It shows that field tomato pathogens (P. phaseoli, F. equiseti, I. lacteus) and research with sequencing of cloned PCR-amplified rDNA saprotrophic species C. cochliodes and Clonostachys sp. A. can be implemented for research of mycobiota and search alternata was detected in most of the studied fruits. C. for new fungal species on the plant, but not for screening coccodes is a widespread tomato pathogen (Belov et al., for dangerous pathogens. 814 Short communication DOI: /10.5513/JCEA01/21.4.2869 Kokaeva et al.: Fungal diversity in tomato (Solanum lycopersicum) leaves and fruits in Russia... Gardes, M., Bruns, T. D. (1993). ITS primers with enhanced specificity CONCLUSIONS for basidiomycetes-application to the identification of mycorrhizae and rusts. Molecular Ecology, 2 (2), 113-118. This research provides new information on the Ivors, K. L., Bartz, F. E., Toda, T., Naito, S., Cubeta, M. A. (2009) First mycobiota of tomato leaves. Several new tomato report of tomato foliar blight caused by Rhizoctonia solani AG-3 basidiospore infection in North America. Phytopathology, 99, S57 pathogens were found. The emergence of new species Kamlesh, Shah, Ramchandra, S. (2017). Eect ff of Culture Filtrate shows the need for continuous improvement of disease of Fusarium equiseti on Tomato Seedling Indices. Biological, control management and adjustments of the lists of plant Environmental and Agricultural Sciences, 2, 45-48. Kokaeva, L. Y., Belosokhov, A. F., Skolotneva E. S., Doeva L. Y., Elansky S. protection products used. N. (2018a). Distribution of Alternaria species on blighted potato and tomato leaves in Russia. Journal of Plant Diseases and Protection, ACKNOWLEDGMENTS 125, 205-212. DOI: https://doi.org/10.1007/s41348-017-0135-3 Kokaeva, L. Y., Berezov, Y. I., Chudinova, E. M., Elansky, S. N. (2018b) The work of L.Y. 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(2019) Leaf-associated microbiomes of graed t ft omato plants. Scientific Reports, 9, 1787. DOI: https://doi.org/10.1038/s41598-018-38344-2 Powered by TCPDF (www.tcpdf.org)

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