Advances in Research on Rape Sclerotia and Resistance Breeding

Yan YUAN, Suping GUO, Guanghuan YANG, Ba DAN

Key Laboratory of Plateau Crop Molecular Breeding, Tibet Agricultural and Animal Husbandry College, Nyingchi 860000, China

Abstract Rapeseed is an important oil crop with high economic value. It can be used not only as edible oil and livestock feed, but also in medicine, industry and tourism. Sclerotium sclerotiorum is a necrotrophic fungal pathogen that harms the yield and quality of rape. This article mainly summarizes the research status of S. sclerotiorum from three aspects: the biological characteristics, infection mode, process and disease resistance breeding of S. sclerotiorum, and summarizes the future research directions of antibacterial sclerotium on rape, to provide reference for future research on sclerotinia.

Key words Rape, Infection with sclerotium, Disease resistance breeding

1 Introduction

Rape is one of the world’s four major oil crops (soybeans, sunflowers, canola, peanuts). Sclerotinia sclerotiorum is a fungal pathogen. Sclerotinia sclerotior is weak, but it has strong saprophyticity. The host range is very wide. There is no specific host[1],and 278 genera can be parasitized in 75 families, more than 400 plants, most of which are dicotyledons[2].Sclerotiumsclerotiorumis one of the main diseases (sclerotia, downy mildew, virus disease) in the main rapeseed producing areas of China. Sclerotium disease causes plant stems and leaves to rot, and the pods die. The onset of near-ground stems will cause rapeseed plants fall or stumble, which in turn affects rapeseed yield.S.sclerotiorumresults in a 10% to 20% reduction in rapeseed yield, and a significant loss in field production of up to 80%[3]. At present, the annual output of the country is about 14 million tons of rapeseed. If the loss is 10% based on the output, the direct economic loss caused by sclerotinia sclerosis alone will be about 7 billion yuan per year. Sclerotinia can also cause the oil content of rapeseed to decrease and the fatty acid composition to change, which seriously affects the quality of rapeseed oil[4]. In recent years, with the continuous improvement of farming practices, the planting density of farmland rapeseed has been increasing, providing conditions for the occurrence ofS.sclerotiorumto a certain extent. At present, prevention and control measures, chemical control, microbial control, and disease-resistant breeding are mainly used for prevention and treatment, among which disease-resistant breeding is the most effective way. Therefore, by exploring the germplasm resources of disease resistance, studying its genetic background, breeding resistant materials through traditional breeding, tissue culture, genetic engineering and other breeding methods is of great significance for improving the yield and quality of rapeseed.

2 Biological characteristics of S. sclerotiorum

S.sclerotiorumis a fungal disease, also known as white rot, stem rot and soft rot.S.sclerotiorum, also known as sclerotium, is a worldwide distribution of phytopathogenic fungi, which belongs to the fungal community, ascomycetes, Aspergillus, Rhizoctonia, Sclerotinia,Sclerotiniagenus, is a facultative parasitic fungus with strong saprophyticity[5]. There are three main forms of existence in the life cycle of Sclerotinia: mycelium, sclerotia and ascospores. The mycelium on the medium and plant body often appears white and brown, the shape is filamentous, with multinuclear and septum, and the hyphae are branched, unable to produce conidia. The hyphae are divided into vegetative hyphae and mycobacteria wire. The sclerotium is a complex structure composed of deposited melanin, which keeps the pathogen viable for a long time under adverse growth conditions and maintains a state of long-term survival. Changing the environmental conditions can cause the sclerotia to germinate to produce mycelium or ascospore. The ascospore plate is born on the sclerotium, often in the shape of a disk, and the central part forms a depression inward, with a handle, and the sub-solid layer is mostly beige, cinnamon or light brown. The color of the sub-layer is slightly lighter than the sub-solid layer, and the base of the handle is dark; the outer capsule is coated with horn cells, and the outer cells are spherical and elliptical. The ascus is nearly cylindrical, the folded ascus is in the form of a stick, consisting of 8 ascospores, and the ascospores are elliptical to nearly fusiform, colorless, single cell[6]. Sclerotia is a mycelial aggregate that plays an important role in the life cycle ofS.sclerotiorum. It not only produces toxic substances, but also maintains the vitality ofS.sclerotiorumin soil. It is the main survival form ofS.sclerotiorum. It is mainly found in soil and diseased bodies and can survive for 8 years in the soil. A large amount of deposited melanin makes the sclerotial nucleus appear black, and melanin plays an important role. On the one hand, it can maintain the water and nutrients inside the sclerotium; on the other hand, it plays an environmental factor that isolates the outside world, so that the fungus can survive the adversity.

The development of sclerotia mainly consists of the following stages: vegetative growth, sclerotia formation, sclerotia growth and sclerotia maturation. During the growth stage of mycelial trophozoites, the mycelium ofS.sclerotiorumgrows to form white villous hyphae; then the sclerotia grows into the growth stage, the villous hyphae grows continuously, and the surface of the mycelium forms a white or light yellow. After the sclerotia grows into the mature stage, the deposition of melanin on the surface of the villous hyphae will gradually harden and form a black sclerotium. At the same time, there will be light yellow droplets seeping out[7]. The main pathogenesis ofS.sclerotiorumis from flowering to maturity. However, in recent years, with the change of farming methods, the pathogenesis ofS.sclerotiorumhas also undergone certain changes. In the rapeseed seedling stage, plants are susceptible[8]. When the seedlings are sick, the base of the stem of the plant will show watery rot, which can cause the plant to stumble. Plants withS.sclerotiorumin flowering stage have light brown lesions on the stems. Later, the light brown lesions gradually become white earth color, and the diseased parts are slightly sunken, which eventually leads to tissue decay, hollow stems and easy peeling of the epidermis. Symptoms of the pods will produce yellow-white lesions, and the severe ones will dry out early. Under wet conditions, white cotton-like mycelium was produced on the surface of the susceptible part, and the inner marrow of the stem of the seriously diseased plant became flocculent, which was mixed with irregularly shaped, black sclerotium lumps[9].

3 Infection method and process of sclerotinia

3.1 Infection methodThere are two ways for sclerotia to invade the host. One is to invade the host directly through the ascospores, and the other is through the hyphae formed by the ascospores and sclerotia. The hyphae can directly invade the host, and the ascospores cannot directly invade. Dye the host. Studies have shown that only whenS.sclerotiorumspores are placed on the host surface with nutrients, it can germinate to form hyphae invading the host, and although some as diffuse substances in the ascospores germinated into the stomata, the mycelium Invasion can only be done through the stratum corneum. The senescent petals of rapeseed can be used as nutrients for germination of ascospores, that is, susceptible petals are the medium of sclerotinia infection[10].

3.2 Pathogen infection processThe specific process of Sclerotinia infecting rapeseed is: the hyphae growing on the surface of rapeseed leaves grows and branches, first, forming the finger-infected pad primordium, and further develops into a dome infestation pad; the top hyphae of the infested pad. An irregular and closely packed bifurcated branch, the apical hyphae form an invasive nail after contacting the host epidermis. The infested nail invades the leaf wax, the stratum corneum, and the epidermal cell wall by the pressure of the applicator and the dissolution of the enzyme on the host surface. After invading the epidermal cells, vesicles are formed, and radial hyphae are formed on the vesicles, and the rapeseed tissue rots as the hyphae expands. The invading hyphae first expand laterally in the epidermal cells, and then form radial hyphae; the radial hyphae can spread between the cells and directly penetrate the cell expansion, leading to decay of the rapeseed tissue[11]. The process of infecting disease-resistant and susceptible rapeseed byS.sclerotiorumis different. Anti-disease can inhibit the growth of hyphae and the formation of adherent cells and infestation mat within 2-3 h of inoculation, and the hyphae can be observed. The protoplasts were squeezed. Within 8 h of inoculation, the infection of the hyphae only stayed on the epidermis of the leaves. For the susceptible varieties, it was obvious that the hyphae had invaded the sponge tissue after 6 h of inoculation, and it was found that calcium oxalate crystals were produced in the epidermis and the palisade tissue. Mycelium has infected the entire leaf tissue at 8 h of inoculation[12].

4 Inheritance of resistance to S. sclerotiorum

Since the fully immunized rapeseed germplasm resources have not been screened, the researchers can only reveal the inheritance of rape resistance by studying the genetic laws of materials with partial resistance. Zhang Yuetal.[13]used 40 pairs of SRAP primers with high polymorphism and clear bands to analyze the genetic diversity of 43 rapeseed materials with good performance in stalk inoculation. It was found that the genetic similarity coefficient was mainly distributed at 0.65-0.77, there is a high genetic similarity, indicating that the genetic difference between the tested rapeseed materials is small, and further genetic screening should be carried out to broaden the existing genetic background of rapeseed. Huang Yongjuetal.[14]used six resistant and susceptible rapeseed varieties from Europe and China as materials to study the genetic attributes and combining ability ofS.sclerotiorumresistance by double-column hybridization and matchstick inoculation. Nuclear disease resistance is mainly controlled by nuclear genes, but there is a certain maternal effect. The nuclear gene is characterized by dominant and additive genes. The resistance ofS.sclerotiorumis partially dominant, and different resistant and susceptible varieties. There is a difference in the degree of resistance dominant. He Kunyanetal.[15]used the 6th generation gene-multi-gene mixed genetic analysis model to study the inheritance of Sclerotinia sclerosis resistance inBrassicanapusL., and found that resistance was not only controlled by two pairs of major genes, but also modified by multiple genes;The disease site is epistatic, and there may be genotype interaction with the environment. Li Lixiaetal.[16]studied in the genetic mechanism of resistance toS.sclerotioruminB.napusfound that the genetic additive and dominant effects of resistance were simultaneously controlled, and the dominant effect was more important than the additive effect. In summary,S.sclerotiorumresistance is mainly controlled by nuclear genes and is a complex quantitative trait controlled by micro-effects and multiple genes. Resistance appears to be partially dominant, additive, and interacts with the environment.

5 Research progress on breeding of rapeseed antibacterial nucleus

5.1 Traditional breedingThe screening of resistance sources is the basic work of disease-resistant breeding. The use of high-resistance varieties for hybridization and selection is an effective way to cultivate resistant varieties. Recurrent selection has been a successful example in rapeseed disease resistance breeding. Jiang Xiaoyanetal.[17]used L135, Zhongshuang 9 as donor parents of antibacterial nuclear disease, respectively, hybridized with the recipient parents 7-5, 7-6, and the hybrid offspring and the corresponding recurrent parents 7-5, 7-6. After repeating backcrossing twice, the selected disease-resistant materials were self-crossed and a new disease-resistant recovery system was developed. Wuetal.[18]used materials containing Ogu sterile cytoplasm, restore gene heterozygosity[S(Rfrf)], and genetic background with Zhongshuang 11 as the female parent, artificial synthetic rapeseed, semi-synthetic rapeseed, the material with excellent resistance in the natural population ofB.napusis the male parent. 8D110-11, 8D112-5, 8D114-2 and 8D117-3 high antibacterial nucleus diseases were selected by molecular marker-assisted selection and recurrent selection. Ogu CMSB.napusreincarnation selection group. Liu Chuietal.[19]used batches ofB.napusand single and double low rapeseed varieties (lines) and their different combinations of hybrids as materials to combine field natural identification with indoor artificial inoculation, and through system identification and research. It is concluded that there are significant differences in resistance to sclerotinia between the varieties (lines) ofB.napus(including double high and single and double low oil), and hybridization and backcrossing with strong disease-resistant parents. Both generations have obvious strong disease-resistance characteristics of partial parents, and strong disease resistance is partially dominant, indicating that there is a difference in disease resistance between genotypes, suggesting that cross breeding or backcrossing of disease-resistant parents in future breeding, and Strengthening the separation, identification and selection in the early generation will increase the possibility of breeding new varieties of single and double low quality rapeseed with strong disease resistance. In the course of the research, a batch of single and double low-yellow rapeseed varieties and selection materials were obtained. Wan Huafangetal.[20]wild-type cabbage C01 with prominent disease resistance as parent material, artificially synthesizedB.napusRB165 with higher resistance than the disease-resistant variety ’Zhongshuang 9, enriching the parent material for disease-resistant breeding Genetic background. Through thorough hybridization, excellent germplasm resources can also be discovered and utilized. Zhao Jianweietal.[21]studied the changes of the new resistant varieties 9629 and YD16 inoculated withS.sclerotiorumafter the hybridization ofB.napusand Songlan with the medium resistant variety Zhongyou 821 and the susceptible variety S-89 as control. It was found that the PPO and PO activities of the distant hybrid lines were higher than those of the control varieties. Li Yuehuaetal.[22]used the F2 population constructed by the high-anti-wild cabbage "C01" and the susceptible cultivation of cabbage "C41" found in the previous stage to locate five QTLs resistant to Sclerotinia disease, using "C01" and cabbage-type rapeseed. The "6Y733" haploid pathway, with "6Y733" as the recurrent parent, transferred the located QTLs locatedon the C9 chromosome to "6Y733", and obtained the cabbage-type rapeseed variety with higher stem resistance than the parent. Wang Haojieetal.[23]used medium-resistantB.napus"Zhongshuang 9" and high-resistance wild cabbage resources for parental hybridization to obtain F1(ACC), F1 continuous use of Zhongshuang 9 back 6 generations, cultivated High resistance material F6.

5.2 Petal-free breedingPetal-free rapeseed is generally obtained by natural mutation and artificial hybridization or mutagenized progeny. The main production pathways are: EMS mutagenesis, interspecific hybridization, interspecific hybridization, and natural mutation. The effect of petal on the growth of rapeseed is mainly due to the following points: (i) the formation, extension and respiration of petal during the flowering process of rapeseed will consume a certain amount of energy;(ii) the rape flower will form a yellow corolla layer on the top of the rapeseed plant, affecting the photosynthetic of rapeseed; (iii) the petal is not only the primary source of infection of the pathogen, but also forms a secondary infection as the petals fall on the leaves. Jamauxetal.[24]observed by scanning electron microscopy that the ascospores only grow on the petals to germinate and form hyphae, and then infest the leaves and stems through the falling of the petals, if the ascospores of the sclerotium fall directly on healthy leaves or stalks. It can’t grow long on the top. Previous studies have pointed out that the pathogens ofS.sclerotiorumare mainly infested by ascospores, while ascospores are highly susceptible to infecting withered petals or old leaves and cannot directly infect healthy stems and leaves. The medium of disease[25]. Chen Yuqingetal.[26]found that during the flowering of rapeseed, the ascospores of the pathogens can spread to various parts of the flower organs. Among the flower organs that are scattered and scattered, the probability of carrying the flowers is the highest. The average rate of the flowering rapeseed is 99.3%, stamens and petals. They were 92.0% and 92.3%, respectively, and the petal-free rapeseed was also higher than the stamen. Under the condition that the number of flowers is equal, the sum of the chances of flowering organs in the petal-free line is 33.0%-36.4%, which is 34.1%. Compared with the control species, the rate of invasiveness between the leaves of the petal-free lines after flowering was significantly lower than that of the control species, and the invasive rate between the plants before maturity was relatively reduced by 32.0%-47.1%. Hu Baochengetal.[27]found in the breeding of sterile lines that the stamens of the sterile line 90A did not develop and the pistil excerpted, which caused the petals to unfold and fall off. The rate of disease and disease index of the sterile line were different. Reduced by 84.46% and 99.57%. Fu Shouzhongetal.[28]reported that the incidence of petal-free varieties was significantly lower than that of petal varieties, and the incidence rate in the field decreased by 80.2%-94.8%, and the petal-free traits were genetically stable. Therefore, petal-free breeding is an important way to breed antibacterial nuclear disease rapeseed varieties. When inoculated by mycelial mass, the disease resistance of the petal-free plants is not superior to that of the petal plants. so, the petal-free material avoids the disease by the morphology, and does not have strong disease resistance by itself. Zhang Jiefuetal.[29]identified 882 rapeseed germplasm resources at home and abroad for resistance toS.sclerotiorum, and found that the growth period of different germplasms is closely related to the resistance to disease resistance. Late maturity germplasm with late flowering period is generally Strong resistance to disease. In the germplasm of the same maturity, the incidence ofS.sclerotiorumand disease index decreased by 30.2% and 38.8%. respectively, and the petal-free plants showed certain disease resistance.

5.3 Tissue cultureTissue culture technology has great potential in the field of breeding. Based on the basic theory of cell pluripotency, it uses a plant’s isolated organ tissues, such as roots, stems, leaves and flowers of plants, to cultivate a kind of complete fertile offspring. technology. It has applications in various disciplines such as botany, agronomy, and medical engineering, especially in crop breeding[30]. Wu Liyouetal.[31]used oxalic acid toxin as a screening agent to treat rape callus, obtained toxin-resistant mutants, and obtained a small amount of regenerated plants after differentiation and culture. The treatment ofinvitroleaves with toxins,invitroleaves of inoculated sclerotinia mycelium, field plants and field disease were identified by several methods. The regenerated plants after mutant differentiation were more resistant than the original lines (2242). The F1 generation material maintains resistance, and the F2 generation material has certain differentiation, but most of them still maintain resistance, among which S11, S21, S23, S24, S25, S26 have obvious resistance. Sex, can be used as a material for screening resistant varieties in the future, broadening the genetic background of breeding materials. Wang Juan[32]constructed two DH line populations by microspore culture technique, and screened new disease-resistant new materials through field disease identification, quality traits and genetic background analysis, and passed the two DH groups obtained. Body leaf inoculation was identified, and 74 and 31 disease-resistant strains were obtained, respectively. Ying Meietal.[33]obtained microspore culture by F1 of (5148 DH×Zhongshuang No. 4 R DH)and F1 of(109 A×Zhongshuang No.4 R DH), and obtained 438 and 172 DH strains, respectively. Then, the two DH populations were identified for disease resistance at the seedling stage and adult stage, and 9 single plants with resistance at the seedling stage and adult stage were obtained in the two DH populations. Huang Jianhuaetal.[34]used Pingyangmycin to mutagenize the haploid shoot tips ofB.napuslines 9985 and 9841, and used oxalic acid as the selection pressure to obtain a batch of strains with higher grass acid resistance than the original varieties body. The disease resistance was identified in the field, and three germplasm materials with significantly improved disease resistance than the disease-resistant varieties were screened out. In summary, the currently selected high-resistance materials can be induced to produce mutants through tissue culture techniques, and then the materials with better disease resistance can be screened.

5.4 Genetic engineeringIn recent years, with the development of genetic engineering technology and the establishment of rapeseed transformation system, more and more scholars have tried to solve the disease resistance problem of rapeseed by means of genetic engineering. Therefore, the application of genetic engineering technology in rapeseed disease resistance breeding The more extensive. By exploiting the disease resistance gene, the target gene can be directly introduced into the desired recipient plant through the vector by genetic engineering technology, and the target gene is specifically expressed in the receptor, so as to achieve the purpose of disease resistance. Genetic engineering technology shortens breeding time compared to traditional breeding methods. Since the fully immunized rapeseed material has not been found so far, and it has been found through research that the resistance ofS.sclerotiorumis a quantitative trait, it is not controlled by a single pair of genes, and there are additive effects and interactions between genes and the environment. Therefore, existing disease resistance genes cannot be directly introduced into disease-resistant materials.S.sclerotiorumbelongs to fungi, the fungal cell wall is composed of chitin, and chitinase can hydrolyze chitin. Therefore, many researchers started with chitinase genes in order to solve the problem of disease resistance from sclerotinia. Lan Haiyanetal.[35]used the Agrobacterium-mediated transformation method to transfer the β-1, 3-glucanase gene of antibacterial nuclear disease and the bivalent plant expression vector of chitinase gene intoB.napus. The test found that the resistance of the genetically modified material was significantly enhanced. Guo Xuelanetal.[36]obtained transgenic plants by introducing corn transposon into the stalk cotyledons ofB.napus. By analyzing the T0 generation and its self-crossing progeny T1, it was found that the transposable element has transposition activity in transgenic rapeseed. Field investigation found that the disease index of sclerotinia sclerosis decreased with the increase of the positive proportion of corn transposon gene Ac in the offspring. The two are linear. Chen Yanetal.[37]transferred the plant protein AP24 gene and glucanase gene in tobacco intoB.napus, and obtained the transgenic bivalent disease resistance rapeseed. The identification showed that the resistance of rapeseed was good. Liu Dan[38]determined the plaque area of transgenic plants by using the transgenic lines of 4-CL overexpressing gene T2 transgenic lines. Liu Feietal.[39]using methods of Agrobacterium tumefaciens transformation of barley oxalate oxidase (Y14203) gene intoB.napus, significantly improved the resistance of rapeseed to Sclerotinia. The reason for the analysis is that oxalate oxidase decomposes oxalic acid into CO2and H2O2, which increases the decomposition rate of oxalic acid and regulates gene expression on the H2O2signal pathway. Sang Xuelianetal.[40]used the Agrobacterium-mediated method to introduce the MSI-99m gene into theB.napuscultivar "Zhongyou 821", and evaluated the resistance of transgenic rapeseed. It was found that the expression of MSI-99m gene inB.napuscould enhance the resistance ofB.napusto Sclerotinia.

6 Problems and prospects

At present, researchers have done a lot of work in the research ofS.sclerotiorum, and cultivated and screened resistant varieties from various aspects, especially in the research of genes, by introducing chitinase gene, corn transposon, Screening resistant varieties by means of disease resistance genes, but it is concluded that the resistance of rapeseed toS.sclerotiorumis a quantitative trait, which is controlled by multiple minor genes, so the single disease-resistant gene will be introduced. The problem of infection of rapeseed byS.sclerotiorumcannot be solved in the susceptible body. Possible directions for future research of sclerotinia are as follows. (i) RNA interference technology can be used to obtain disease-resistant plants, and the key genes ofS.sclerotiorumare linked together to construct short hairpin RNA, which is introduced into rapeseed by vector, and short hairpin RNA is unwound. The enzyme unwinds to form the sense strand and the antisense strand, and the antisense strand forms a silent complex structure with some enzymes in the cell (ligase, endonuclease, exonuclease,etc.), and antisense whenS.sclerotioruminfecting the strands are complementary to the sclerotiorum mRNA and cleave the mRNA, which limits the gene expression ofS.sclerotiorumand thus acts as a disease resistance. (ii) According to scholars research, most of theS.sclerotioruminfects dicotyledonous plants and infects other plants less.By studying the mechanism of infection, we can find out the cause of infection by the difference between dicotyledonous plants and other kinds of plants. Change the susceptible structure to prevent sclerotinia. (iii) The lodging of rapeseed and the occurrence ofS.sclerotioriumshow mutual promotion. Thus, the lodging of rapeseed reduces the wind permeability in the field, the light is weakened, the humidity is increased, which is conducive to the occurrence ofS.sclerotiorum; occurrence will cause the cane stem to hollow up. Therefore, the lodging resistance of rapeseed should be considered when breeding resistant varieties.