HUE UNIVERSITY: Nghiên cứu giông hồ tiêu kháng Meloidogyne incognita bằng chỉ thi phân tử

HUE UNIVERSITY INSTITUTE OF BIOTECHNOLOGY ***** SONEXAY RASPHONE RESEARCH ON PEPPER VARIETIES (Piper spp.) RESISTANCE TO Meloidogyne incognita BY MOLECULAR MARKERS IN VIETNAM Major: Biology ID: 9420101 SUMMARY OF DOCTORAL THESIS IN BIOLOGY Scientific supervisors: ASSOCIATE PROFESSOR PH.D TRUONG THI HONG HAI PH.D NGUYEN QUANG CO HUE, 2024 PREAMBLE 1. The urgency of the Thesis Pepper (Piper spp.) is a crop with great economic value in Vietnam. In 2022, the pepper growing area across the country will be 131.8 thousand hectares, and exports will reach 228.7 thousand tons, with total export turnover increasing by 3. Vietnam accounts for 40% of output and 60% of the global pepper market share, while maintaining the number one position in the world in terms of production and export (Vietnambiz, 2023).

In Vietnam, pepper varieties being grown popularly in production can be classified into three groups: small-leaved pepper including Se, Se Dat Do, Vinh Linh, Tieu Son, Di Linh, Phu Quoc, and Nam Vang; Medium leaf pepper is usually imported from Madagascar, India and, Indonesia such as Lada Belangtoeng, Karimunda, Kuching and Panniyur; Large-leaf peppers include Se Mo and Trau Dat varieties, among which the three most commonly grown groups are Lada Belangtoeng (Sung, 2001). In recent years, due to climate change combined with the development of pepper trees beyond the orientation and not according to the plan, the situation of pests and diseases on pepper plants has appeared more and more, including the two most serious diseases the fast-dead and the slow-dead. According to a report by the Plant Protection Department at the beginning of 2019, the area of pepper trees that died was more than 10 thousand hectares, mainly due to harmful diseases, in which the disease died quickly due to Phytophthora fungus and the disease died slowly caused by the fungus Phytophthora. Meloidogyne incognita is considered the most harmful disease for pepper plants.

According to research and experiences in pepper cultivation in the world and in Vietnam, the control of harmful nematodes on pepper plants by chemical drugs is very ineffective, costly, and pollutes the environment (Youssef & El-Nagdi, 2021). In addition, the use of crop rotation techniques and the use of Mycorrhizal arbuscular fungi (Mandou et al., 2023) or the use of biological products to control nematodes have also been published (Lockett et al., 2000; Xuyen, 2000; Dong & Zhang, 2006; Anwar & Rashid, 2007; Caillaud et al., 2008; Claudius-Cole et al., 2010; Sowley et al., 2014; El-Nagdi & Youssef, 2015; El-Nagdi et al., 2019; Mhatre et al., 2019; Thuy et al., 2019; Youssef & El-Nagdi, 2021; Lawal et al., 2022; Burns et al., 2023; Bhat et al. The use of parasites in nematode control has also been studied (Rahanandeh, 2012; Mukhta & Pervaz, 2013; Mukhta et al., 2013; Saad et al. However, the most effective method to control nematodes is the use of resistant pepper varieties (Eapen & Pandey, 2018; Ngoc et al.

Therefore, the research and breeding of pepper resistant to nematodes is very necessary for current and future pepper production. Local varieties are used in breeding programs because of their potential to carry genes for resistance to plant diseases and pests, as well as providing a source of genetic diversity for plant breeding (Nas et al. However, as pepper is a perennial plant, it takes a lot of time and effort to select and create new varieties according to traditional methods to select varieties with desired traits, especially tolerance traits. adapt to the changing environmental conditions.

There have been many research projects on breeding pepper plants with high quality, efficiency, and productivity. In particular, the South American wild pepper (Piper colubrinum) and the betel nut (Piper betle) are quite resistant to the fungus Phytophthora capsici and the nematode Meloidogyne incognita (Hien et al., 2019) and have good compatibility when grafted with Vinh Linh pepper (Piper Nigrum) (Ngoc et al. Nowadays, with the development of the biotechnology industry, the work of selecting and creating new plant varieties has become more convenient and easier, especially using molecular marker techniques in breeding that can quickly and accurately select desired traits, shorten time, increase yield, select and create genetically accurate target varieties as well as save effort (compared to traditional breeding and selection). So, “Research on pepper varieties (Piper spp.) resistance to Meloidogyne incognita by molecular markers in Vietnam” is urgent to select nematode-resistant varieties and develop solutions for stable and sustainable pepper production.

In this study, waterlogging-tolerant black pepper varieties were also selected to select varieties suitable for the frequently flooded conditions of Thua Thien Hue Province. Objectives of the study Overall objective Research on pepper varieties (Piper spp.) resistance to Meloidogyne incognita by molecular markers in Vietnam. Details objective Evaluation of genetic diversity of pepper groups collected in Vietnam Selection of some pepper lines/varieties that are resistant to root knot nematode (M. incognita) and tolerant to waterlogging The development of molecular markers helps identify nematode resistance of pepper strains/varieties.

Evaluation of flowering characteristics of some pepper lines/varieties of P. and the possibility of crossbreeding with 2 nematode-resistant P. divaricatum to create new pepper lines/varieties for Vietnam Selection of some good conjugative graft combinations that are resistant to nematodes Evaluation of the growth and development ability of a nematode- resistant pepper graft combination under greenhouse conditions. New points of the thesis Successfully identified and evaluated genetic diversity using morphology and molecular markers of pepper lines/varieties collected in Vietnam Selected a pepper line/cultivar of Piper hancei (HUIB_PH30) and a pepper line/cultivar of Piper devaricatum (HUIB_PD36) that are resistant to gall nematode M.

incognita and have good waterlogging tolerance. The molecular marker SCAR 30 - 360F1R2 associated with nematode resistance of pepper plants has been developed. Flowering characteristics of P. and the possibility of weak hybridization between P.

divaricatum were evaluated. Selected two pepper graft combinations (HUIB_PH30 - Vinh Linh and HUIB_PD36 - Vinh Linh) that are well compatible, resistant to gall nematodes and grow and develop well in greenhouse conditions CHAPTER 1 DOCUMENTARY OVERVIEW 1. Theoretical basis of the research 1. Synopsis of Nematodes 1.

Introduction to nematodes The nodule nematode, of the genus Meloidogyne (Trinh et al., 2019), family Meloidogynidae, order Tylenchida (Kofoid & White, 1919), is one of the main pathogens found in many different plant species (Sikandan et al., 2020; Yang et al. Root-knot nematode (Meloidogyne spp.) is a pathogen affecting the quality and yield of pepper varieties, and M. incognita is economically one of the most important plant parasitic nematodes. worldwide due to its increasing geographical distribution, wide host range, and pathogenicity (Nas et al.

Classification of nematodes In Vietnam, Meloidogyne spp., Rotylenchulus reniformis, Ditylenchus ausafi, and Aphelenchus avenae are five plant parasitic nematodes found in all studied provinces. Meloidogyne spp, is the common taxon found and all Meloidogyne is recognized as M. The harmful effects of nematodes Meloidogyne is known to be one of the major pests of vegetable, medicinal and other crops. In pepper, this group of nematodes is a major cause of the disease of “slow death”, yellowing of leaves, and reduced yield of pepper (Quyen et al.

Nematodes cause a 15% annual crop loss, estimated at $100-157 billion worldwide (Abd-Elgawad & Askary, 2015). In the pepper-growing countries of Southeast Asia, Meloidogyne spp causes losses of up to 16% (Sasser, 1979). Measures to treat nematodes Various synthetic nematodes have been used to control nematodes, however, most pesticides have been removed from the market due to off- target effects and effects on human health and the environment. Trichoderma, mycorrhizal and endophytic fungi are the main filamentous fungi used to confer nematode resistance.

They can reduce damage caused by parasitic nematodes on plants by producing enzymes that break down, antibiotic, paralyze, and parasitize. In addition, many species of fungi with the ability to kill nematodes have been tested such as Dactylella oviparasitica, Arthrobotrys oligospore, Monacrosporium gepgyropagum, Verticillium chlamydosporium. Summary of black pepper 1. About black pepper Pepper (Piper nigrum L.) is a perennial climbing plant belonging to the Piperaceace family (Bui et al., 2017), originating from India, then introduced to tropical countries in Asia and America such as Indonesia, Vietnam, Brazil, etc.

Pepper is often called the king of the most used spices in the world (Khew et al., 2020; Dongare et al., 2023) and it has become familiar in people's daily dishes. Besides, pepper is also used in medicine to treat many diseases such as flu, congestion, arthritis,. (Wang et al. The role and effects of black pepper Pepper is a perennial crop with high economic value.

Pepper is used as a spice, in the flavoring industry, in medicine, and as an insecticide (Hoa, 2001). Morphological characteristics of black pepper Pepper roots include 3-6 taproots and a bunch of auxiliary roots below the ground, on the stem with lizard roots (root attachments). Pepper is a flexible herbaceous plant that is divided into several segments, each with a single, heart-shaped, alternate leaf. In the leaf axils, there are dormant sprouts that can arise into twigs, eel branches, and evil branches (left branches) depending on the stage of development.

Pepper plants flower in the form of spike-shaped flowers, dangling, 7-12 cm long, depending on pepper varieties and care conditions. On the 4 flower spike, there are an average of 20-60 flowers arranged in a spiral, the pilot bisexual or unisexual. The fruit is a nut, without a stem, bearing 1 spherical seed. From full flowering to fruit, ripening lasts 7-10 months.

Distribution of pepper Pepper originated in India, then imported to tropical countries such as Brazil, Indonesia, Malaysia, Thailand, Sri Lanka, and Vietnam. Today, although pepper is found in almost all tropical countries, the main production areas are concentrated in a few countries of South Asia, Southeast Asia, and Brazil. Pepper is widely grown in many places: India, Brazil, Indonesia, Malaysia, Sri Lanka, Vietnam, and China (Xuyen et al. Pepper varieties in use In 2021, Thuy et al.

collected 33 pepper samples from the Southeast, Phu Quoc, and Central Highlands regions, including Vinh Linh, Sri Lanka, Brazil, India, and Se pepper varieties. Based on morphology, pepper can be divided into two lines: small-leaved small- leaved pepper includes: Se Dak Lak, Phu Quoc and Vinh Linh pepper collected in the Southeast, and Central Highlands. Large-leaved pepper varieties include Brazilian pepper grown in Binh Duong, Indian pepper grown in Dong Nai, and Sri Lankan pepper grown in Gia Lai and Dong Nai. Breeding methods of pepper varieties To improve the characteristics of pepper varieties and increase resistance to pests and diseases, cross-breeding methods are essential, to create new varieties, and increase the diversity and richness of pepper genetic resources.

There are two methods, natural hybridization, and artificial hybridization. Breeding through free pollination (natural hybridization) is increasingly popular and gives very good yields. Grafting methods applied on black pepper Grafting of pepper (Piper nigrum) on resistant root-stocks of P. colubrinum is a widely accepted technique for the management of Phytophthora diseases.

To evaluate the effect of variety and season on graft recovery, a preliminary study was performed. In which the lateral shoots of eight varieties of P. nigrum were grafted on P. colubrinum root-stock.

The results show that regardless of variety, February and March are the best times for grafting (Vanaja et al. Definition of a molecular marker Molecular markers, or DNA markers, are markers that are located only near or associated with genes and have little or no effect on phenotype. DNA markers are changes in DNA and are divided into several types based on different methods and techniques for identifying 5 polymorphisms (Thanh, 2014). Types of Molecular Markers Current markers include: Restriction Fragment Length Polymorphism (RFLP), Short Tandem Repeat (STR), Variable Number of Tandem Repeat (VNTR), Single-Strand Conformation Polymorphism (SSCP), Sequences-tagged sites (STS), Random Amplified Polymorphic DNA (RAPD), Single Nucleotide Polymorphism (SND), Restriction Fragment Length Polymorphism (RFLD), Sequence tagged microsatellite site (STMS), DNA amplification fingerprinting (DAF), Expressed Sequence Tags (EST) (Adams et al.