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GomzhinaМ.М., Gannibal Ph. B./Plant Protection News, 2020, 103(3), p. 207-210
диагностики фитопатогенных грибов является ПЦР с видоспецифичными праймерами. Такой метод позволяет проводить высокоточную детекцию и идентификацию целевых объектов. Цель данной работы заключалась в разработке видоспецифичных олигонуклеотидных праймеров, избирательно амплифицирующих ДНК гриба P. lindquistii. Праймеры LepliF2/LepliR2, разработанные на основе анализа ITS локуса, показали стабильную амплификацию ДНК целевого гриба при отсутствии кросс-реакции с другими видами грибов. Эти праймеры могут быть рекомендованы для проведения экспресс-диагностики возбудителя фомоза подсолнечника. Данная работа представляет собой первую разработку в области молекулярной экспресс-диагностики этого патогена.
Ключевые слова: молекулярная диагностика, фомоз, чёрная стеблевая пятнистость, подсолнечник Поступила в редакцию: 06.05.2020 Принята к печати: 19.08.2020
OECD+WoS: 4.01+AM (Agronomy) https://doi.org/10.31993/2308-6459-2020-103-3-4998
Short communication
FUNGAL PATHOGENS OF TOMATO IN SOUTH-WESTERN RUSSIA (KRASNODAR TERRITORY)
E.M. Chudinova1, T.A. Shkunkova1, S.N. Elansky12*
1 Peoples'Friendship University of Russia, Moscow, Russia 2 Moscow Lomonosov State University, Moscow, Russia
*corresponding author, e-mail: snelansky@gmail.com
During a study of fungal diseases of tomato in the South of Russia (Krasnodar Territory) 56 fungal isolates associated with tomato fruits were obtained. Most of them belonged to the species Alternaria alternata. Alternaria solani, Fusarium equiseti, Phomopsisphaseoli, Chaetomium cochliodes, Clonostachys sp., Irpex lacteus, Colletotrichum coccodes were also identified. Laboratory experiments revealed that Clonostachys sp., C. cochliodes, P. phaseoli, I. lacteus, and F. equiseti developed well on the fruit's slices. Fusarium equiseti was the only species that can penetrate the tomato through epidermis and infect entire fruit. The most effective fungicide against F. equiseti was difenoconazole (EC50 = 0.08 mg/L); pencycuron was also effective (EC50 = 32.5 mg/L). Thiabendazole completely inhibited the growth of F. equiseti at the concentration 100 mg/L (EC50 = 47 mg/L).
Keywords: fungicides, tomato diseases, Fusarium equiseti, Phomopsis phaseoli, Chaetomium cochliodes, Clonostachys sp., Irpex lacteus, Alternaria solani
Received: 09.04.2020 Accepted: 22.07.2020
Introduction
Climatic conditions allow the cultivation of tomato in open ground in the southern regions of Russia. In the Krasnodar Territory (2018) farmers grow tomato in open fields on an area of 750 hectares; the total yield is about 9 thousand tons (ab-centre.ru, small private gardens and greenhouses are not accounted). When grown in open ground, tomatoes are severely affected by diseases and pests. The most common diseases in the south of Russia and adjacent countries are late blight (caused by Phytophthora infestans (Mont.) de Baiy), early blight (Alternaria spp.), Septoria leaf spot (Septoria lycopersici Mart.), Fusarium rot (Fusarium sp.), root and stem rot (Pythium ultimum Trow), powdery mildew (Erysiphe communis (Wallr.) Schltdl., Oidium lycopersici Cooke & Massee), white rot (Sclerotinia sclerotiorum (Lib.) de Baiy),
gray mold (Botrytis cinerea Pers.), leaf mold (Fulvia fulva (Cooke) Cif. = Cladosporium fulvum), black tomato fruit rot (Remotididymella destructiva (Plowr.) Valenz.-Lopez, Cano, Crous, Guarro & Stchigel = Phoma destructiva) (Agaev et al., 2014).
In addition to the aforementioned widespread phytopathogenic microorganisms, new ones are currently appearing. They can cause diseases similar in symptoms. These microorganisms may differ in pathogenicity and resistance to fungicides. The use of effective fungicide preparations is the basis of high-quality tomato protection. That is impossible without the monitoring of tomato pathogens. The aim of our work was to analyze tomato fungal pathogens to search for new species atypical for Southern Russia.
Materials and Methods
The paper represents the results of a study of mycobiota were many plants with lesions caused by insects consequently associated with affected tomato fruits in two studied fields of colonized by bacteria and fungi, as well as plants with fungal, the Krasnodar Territory (Slavyansk-na-Kubani district). There bacterial damage, mixed lesions, and lesions resulting from
sunburn. For the analysis of fungal pathogens, fruits with brown spots or whitish mycelium without punctures of the surface caused by insects were selected. In each case one fruit per plant was taken. Fruits were washed carefully, and surface was sterilized with 70 % alcohol. Their slices were placed in moist chambers. Mycelium or spores were taken from alive tissue using needle under the microscope and placed on a Petri dish with wort agar mixed with penicillin. Further, axenic cultures of fungi were analyzed according to cultural-morphological characteristics. To confirm the results of cultural-morphological identification of species, sequencing of species-specific DNA region (ITS1-5.8S-ITS2, primers ITS5-ITS4, White et al., 1990) was performed for all isolates except small-spores Alternaría.
Pathogenicity tests were conducted on symptomless, detached green tomato fruits, with surface sterilized using ethanol (70 %) and on slices of these fruits. Sterilized fruits were washed in three changes of distilled water. Agar plugs with fungal mycelium was placed in the center of the slice or on the surface of the fruit. Control fruit or slice was inoculated
Results and Discussion
with a small piece of agar only. The fruits were then incubated in a plastic container at 23 °C with wet paper placed on the bottom. Slices were incubated at the same temperature in Petri dishes on the glass lying on the wet paper. Tested fruits and slices were examined for mycelium development for 7 days after inoculation.
Estimation of fungicidal activity was carried out on Petri dishes with different concentrations of the studied fungicides. A block of colonized agar was placed in the center of Petri dish with hard oat medium of four gradually increasing concentrations of active compound: 0.1; 1.0; 10.0 and 100.0 mg/l. The medium without the fungicide was used as a control. Two perpendicular diameters of each colony were measured when diameter of control colony was 70-80 % from radial size of Petri dish. After the measurements average diameter for each isolate was calculated. The effective inhibitory concentration EC50, i.e. the concentration of a fungicide in the medium needed to reduce the radial growth of a colony by half in comparison to fungicide-free control, was determined.
During this study 56 fungal isolates were obtained. The vast majority (44 isolates) belonged to the species Alternaría alternata (Fr.) Keissl. Other species, such as Alternaría solaní Sorauer, Colletotrichum coccodes (Wallr.) S. Hughes, Fusarium equiseti (Corda) Sacc., Phomopsisphaseoli (Desm.) Sacc., Chaetomium cochliodes Palliser, Clonostachys sp. and Irpex lacteus (Fr.) Fr. were also isolated (table 1).
Table 1. Fungal species, isolated from tomato fruits
Species name Number GenBank accession
of strains number
Alternaría alternata 44 Not tested
Alternaría solaní 2 KY496637*
Colletotrichum coccodes 2 MT292616*
Fusarium equiseti 1 MT588081
Phomopsis phaseoli 2 MH412692*
Chaetomium cochliodes 3 MT279444*
Clonostachys sp. 1 MT588112
Irpex lacteus 1 MT276332
* - sequences of all strains were identical
Fusarium equiseti is widespread on tomato in Asian countries (Akbar et al., 2018), but its distribution in Russia has not been studied. Phomopsis phaseoli is one of the common pathogens of soybeans; this fungus was first discovered on tomato (Elansky et al., 2019). The basidiomycete I. lacteus is a wood white rot fungus, which has not been recorded as tomato pathogen. Soil saprotrophic fungi Chaetomium cochliodes Palliser and Clonostachys spp. form antagonistic relationship Table 2. The diameter of the colonies of F. equiseti
with many soil microorganisms. One of Chaetomium species is used in the commercial preparation of Ketomium, which inhibits the growth of pathogens of many significant crops, including tomato (Soytong et al., 2001). Strains of the genus Clonostachys are widely used in biotechnological applications (Borges et al., 2015).
Some isolated fungal species had never been typical tomato pathogens in Russia. Since these fungi were isolated from affected tomato fruits, we evaluated their ability to develop on tomato fruits and slices in a moist chamber. According to our experiments, Clonostachys sp., C. cochliodes, P. phaseoli, and I. lacteus were not able to penetrate the tomato epidermis and infect fruits, but they developed well on fruits' cuttings. On day 7 after infection with Clonostachys sp., a lesion of 18±2 mm was formed on tomato slices (average diameter for 3 tested slices ± standard deviation). Inoculation with other fungal species was also resulted in lesions: 15±3 mm (C. cochliodes), 25±3 mm (P phaseoli), 29±4 mm (I. lacteus). Apparently, these fungi can parasitize on tomato fruits when a crack occurs on their surface. Fusarium equiseti showed high aggressiveness in slices test, after 7 days the tomato slices were completely braided with its hyphae. Fusarium equiseti was the only tested pathogen that can infect the tomato fruits through the epidermis.
In the present study F. equiseti was first discovered on tomato in Russia. We tested its susceptibility to the following fungicides: difenoconazole (preparation Score), thiabendazole (Tecto) and pencycuron (Prestige) (table 2). The most effective fungicide was difenoconazole (EC50 = 0.08 mg/L). This drug
in Petri dishes with medium containing fungicides
Fungicide Colony d 0 íameter* at differ 0.1 ent active compo 1 und concentratioi 10 ns (mg/L) 100 EC50**, mg/L
Diphenoconazole Thiabendazole Pencycuron 45±2 47±2 47±2 20.5±1 Not tested Not tested 6.5±0.5 42±5 41±5 4±1 36±4 26±3 Not tested 0 10±1 0.08 47 32.5
* - average diameter for 3 tested Petri plates (mm) ± standard deviation,
** EC50 - the concentration of fungicide (active ingredient) in the medium needed to reduce the radial growth of a colony by half in comparison to fungicide-free control.
is used for treatment of vegetative tomato plants against early blight. Thiabendazole is recommended for sterilization of storages. Our data showed that at a concentration of 100 mg/L it completely inhibits the growth of F. equiseti. Pencycuron is effective against F. equiseti (EC50 = 32.5 mg/L) and can be recommended for the treatment of tomato seeds (Catalog..., 2020).
Our research provided new information on the mycobiota of tomato fruits in Southern Russia, the main potato producing region of the country. Several new affected tomato fruits with fungal species were found. Detection of new pathogens showed the need for disease monitoring and optimization of disease control management.
The research is supported by the RUDN University Program 5-100.
References
Elansky SN, Shkunkova TA, Chudinova EM, Pakina EN, Kokaeva LY, Alexandrova AV, Krutyakov YA (2020) First report of Phomopsis phaseoli on tomato. J Plant Pathol 102:263-264. https://doi.org/10.1007/s42161-019-00403-6 Soytong K, Kanokmedhakul S, Kuknogviriyapa V, Isobe M (2001) Application of Chaetomium species (Ketomium) as a new broad-spectrum biological fungicide for plant disease control: A review article. Fungal Diversity 7:1-15 White TJ, Bruns T, Lee SJWT, Taylor JW (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. PCR Protoc Guide Methods Appl 18(1):315-322
Agaev DT Bolezni tomata v fermerskikh hozyaistvakh [Farm Tomato Diseases] (2014) Zaschita i karantin rasteniy 9:3833 (In Russian)
Akbar A, Hussain S, Ullah K, Fahim M, Ali GS (2018) Detection, virulence and genetic diversity of Fusarium species infecting tomato in Northern Pakistan. PLoS One 13(9):e0203613. Doi: 10.1371/journal.pone.0203613
Borges AV, Saraiva RM, Maffia LA (2015) Biocontrol of gray mold in tomato plants by Clonostachys rosea. Trop. plant pathol. 40:71-76. https://doi.org/10.1007/ s40858-015-0010-3
Catalog of pesticides and agrochemicals approved for usage on the territory of Russian Federation. Part 1. Pesticides. (2020). M.: Ministry of Agriculture. 283 p.
Вестник защиты растений, 2020, 103(3), с. 210-212
OECD+WoS: 4.01+AM (Agronomy) https://doi.org/10.31993/2308-6459-2020-103-3-4998
Краткое сообщение
ГРИБНЫЕ ПАТОГЕНЫ ТОМАТА НА ЮГО-ЗАПАДЕ РОССИИ (КРАСНОДАРСКИЙ КРАЙ)
Е.М. Чудинова1, Т.А. Шкункова1, С.Н. Еланский1,2* 1 Российский университет дружбы народов, Москва, Россия 2 Московский государственный университет имени М.В. Ломоносова, Москва, Россия
* ответственный за переписку, e-mail: snelansky@gmail.com
При изучении грибных болезней томата в Краснодарском крае из пораженных плодов были выделены в чистую культуру 56 штаммов грибов. При анализе видовой принадлежности коллекционных изолятов культурально-морфологическими и молекулярными методами оказалось, что большая их часть принадлежала виду Alternaria alternata. Также были идентифицированы Alternaria solani, Fusarium equiseti, Phomopsis phaseoli, Chaetomium cochliodes, Clonostachys sp., Irpex lacteus, Colletotrichum coccodes. Лабораторные эксперименты по заражению ломтиков плодов томата показали, что Clonostachys sp., C. rnchliodes, P. phaseoli, I. lacteus, F. equiseti способны их успешно заражать. F. equiseti оказался единственным видом из исследованных, способным заражать неповрежденные плоды, проникая через эпидермис. Оценка восприимчивости F. equiseti к фунгицидам выявила, что наибольшей эффективностью отличался дифеноконазол (EC50 = 0.08 mg/l). Пенцикурон также показал хорошую эффективность (EC50 32.5 mg/l). Тиабендазол полностью ингибировал рост колонии F. equiseti при концентрации 100 mg/l (EC50 = 47 mg/l).
Ключевые слова: фунгициды, болезни томата, Fusarium equiseti, Phomopsis phaseoli, Chaetomium cochliodes, Clonostachys sp., Irpex lacteus, Alternaria solani
Поступила в редакцию: 09.04.2020
Принята к печати: 22.07.2020
