Utilization Situation and Prospect of Gene xa5 Against Pathotype V of Rice Bacterial Blight

Taihui CHENG　Shen CHEN　Jianyuan YANG　Xiaoyuan ZHU　Shengyuan WU　Qijin HONG　Liexian ZENG

Abstract In recent years， the strong virulence pathotype V of rice bacterial blight grew up quickly in Southern China， which has become a major population and spread to the rice regions of Jiangsu and Zhejiang provinces in Southern China. Since pathotype V caused serious bacterial blight disease in rice production regions， it is urgent to breed and promote resistant varieties against pathotype V. The most economic and effective measure to control rice bacterial blight is to breed resistant cultivars for widely planting using resistance genes. The Institute of Plant Protection， Guangdong Academy of Agricultural Sciences， and Agricultural Science Research Institute of Panyu District of Guangzhou used IRBB5 carrying the recessive xa5 gene from IRRI that is resistant to pathotype V to breed resistant varieties with rice blast resistance source， through hybridization， multiple cross， pedigree selection and synchronous resistance evaluation. We successfully bred series of new resistant rice varieties such as Baijiangzhan， Baijingzhan and Baisizhan， which showed resistance to strong virulence pathotype V （grade 1-3） of bacterial blight and rice blast （mediate to high resistance）， good grain quality （level 3 of rice quality of Guangdong）， and yield equivalent to major cultivars （compared with region trial control cultivars of Guangdong）. These new resistant varieties were promoted and planted in the strong virulence pathotype V region along the west coast of Guangdong， which showed favorable superiority and wide application prospect in controlling rice bacterial blight with resistance varieties.

Key words Rice; xa5 gene; Pathotype V of rice bacterial blight; Breeding for disease resistance

Received： May 23， 2020  Accepted： July 29， 2020

Supported by Special Fund of Agro-scientific Research in Public Interest （201303015）; Earmarked Fund for China Agriculture Research System （CARS-01-24; 2019KJ105）; Science and Technology Planning Project of Guangdong Province （2015A020210081）; Special Fund for Science and Technology Innovation Strategy of Guangdong Academy of Agricultural Sciences （R2016PY-JX005， R2016PY-QY009）.

Taihui CHENG （1973-）， male， P. R. China， senior agronomist， devoted to research about rice genetics and breeding.

*Corresponding author. E-mail： 2244642820@qq.com.

Rice is the most important food crop， and 50% of the worlds population and more than 65% of Chinas population rely on rice as the staple food. Chinas rice planting area accounts for about 30% of the countrys total grain crop planting area， and the output has reached 40% of the total grain output. The sown area and total output rank first among grain crops. Therefore， rice production is vital to the food security of China and the world[1-2]. Rice production often faces threats of various diseases and pests to varying degrees. Among them， bacterial blight caused by the Gram-negative bacterium Xanthomonas oryzae pv. oryzae （Xoo） is a bacterial disease causing most serious damage to rice production. The pathogen invades mainly from rice wounds and expands along the leaf veins， and grows and multiplies in the vascular bundles. After the occurrence in rice， the leaves wither， which affects photosynthesis. As a result， the rice does not set seeds or the thousand-grain weight is reduced， and the yield is generally reduced by 20%-30%， or 80%-100% in severe cases[3-7]. Bacterial blight is also one of the three major diseases in rice production in China （rice blast， bacterial blight， and sheath blight）. It occurs widely and is distributed in rice areas in all provinces except Xinjiang. The high temperature and humidity in the coastal rice regions of South China is more conducive to the occurrence and prevalence of bacterial blight， and the disease is particularly harmful to the late-season rice in the typhoon season. Generally， from the perspective of rice type， bacterial blight is severer in indica rice than in japonica rice， severer in sticky rice than in glutinous rice， and severer in hybrid rice than in conventional rice[8-11].

X. oryzae pv. oryzae （Xoo） has pathogenic differentiation. In the late 1980s， a virulent pathotype V appeared in Guangdong Province， and gradually rose and developed. None of the main disease-resistant varieties containing the Xa4 gene and even all varieties planted in Guangdong at that time could resist the pathotype. Theory and production practice have proved that the most economical and effective measure to prevent and control bacterial blight is to breed and promote disease-resistant varieties[11-12]. The Institute of Plant Protection， Guangdong Academy of Agricultural Sciences， and Agricultural Science Research Institute of Panyu District of Guangzhou used IRBB5 carrying the recessive xa5 gene from IRRI that resists pathotype V to breed resistant varieties with rice blast resistance source， and obtained the series of new rice resistance varieties such as Baixiangzhan， Baijingzhan and Baisizhan， which showed resistance to strong virulence pathotype V of bacterial blight and rice blast （mediate to high resistance）， good grain quality， and yield equivalent to major cultivars （compared with region trial control cultivars of Guangdong）. These varieties were planted in the diseased area and achieved obvious prevention and control effects. In this study， the epidemic trend of the strong virulence pathotype V of rice bacterial blight in recent years and the utilization status and prospects of the xa5 gene resistant to the strong virulence pathotype V of rice bacterial blight were overviewed， providing a reference for the selection and production of resistance genes for the breeding of varieties with resistance to rice bacterial blight and the prevention of the spread and expansion of the strong virulence pathotype V of rice bacterial blight in production， effective control of rice bacterial blight， and increase of rice yield.

Pathogenic Differentiation of Rice Bacterial Blight and the Epidemic Trend of the Strong Virulence Pathotype V in Recent Years

Pathotype of X. oryzae pv. oryzae

X. oryzae pv. oryzae has obvious pathogenic differentiation， forming different pathotypes. The resistance to X. oryzae pv. oryzae in China was identified as 9 pathotypes， SRRRR （type I）， SSRRR （type II）， SSSRR （type III）， SSSSR （type IV）， SSRRS （type V）， SRSRR （type VI）， SRSSR （type VII）， RRRSR （type VIII） and SSSSS （type IX） in the host Jingang 30， Tetepu， Nangeng 15， Java 14 and IR26[17] （Table 1）.

Epidemic trend of the strong virulence pathotype V in recent years

Pathotype V is a bacterial strain with a broad spectrum of pathogenicity， strong toxicity， and great harm. Our research team was first discovered in Guangdong at the end of the 20th century. This pathotype can seriously infect the internationally identified variety IR26， and none of the main disease-resistant varieties containing the Xa4 gene in Guangdong and even all varieties planted in production at that time could resist it. According to our monitoring of the occurrence of rice bacterial blight in Guangdong from 2014 to 2017， pathotype V showed the highest frequency of occurrence for 4 consecutive years， and had risen to the dominant pathotype in Guangdong （Table 2）. What is more serious is that this pathotype has been extended to the rice areas of the Yangtze River Basin such as Zhejiang and Jiangsu[18-19]， which poses a great threat to the safety of rice production in China.

Variety Resources Resistant to the Strong Virulence Pathotype V of Rice Bacterial Blight and the Genetic Characteristics of the Main Resistance Genes

Gene Resources resistant to the strong virulence pathotype V of rice bacterial blight

There were obvious differences in the resistance of different resistance genes and rice varieties to different pathotypes of bacterial blight. Up to now， there are 45 rice resistance genes of bacterial blight reported at home and abroad[20]， but there are few genes resistant to the strong virulence pathotype V of bacterial blight. Zeng et al.[21] identified the resistance of the international rice germplasm resources against the main bacterial strains of rice bacterial blight in southern China， and selected from 1 592 germplasm resources only four key resistant varieties， BG1222[Xa34（t）]， IRBB5（xa5）， IRBB7（Xa7） and IRBB203（Xa3）， which are resistant or highly resistant to the strong virulence pathotype V of bacterial blight in southern China and have good economic traits. These disease-resistant resources lay a good foundation for the breeding of disease-resistant varieties.

Genetic characteristics of the strong virulence pathotype V of rice bacterial blight

The resistance genetic analysis shows that the resistance of BG1222 and IRBB5 to pathotype V is dominated by a pair of recessive genes; and the resistance of IRBB7 and IRBB203 to pathotype V is controlled by a pair of dominant genes， and IRBB5， IRBB203 and IRBB7 are near-isogenic lines， of which the known resistance genes are， respectively， xa5， Xa7 and Xa3， among which xa5 is a recessive gene， and Xa7 and Xa3 are dominant genes. The resistance of BG1222 to pathotype V is dominated by a pair of recessive genes， and the allelic determination results of the resistance genes of the varieties IR1545-339-2-2 and IRBB5 that carry the xa5 resistance gene show that the resistance gene carried by BG1222 is not allelic to xa5 and is inherited independently. Chen et al.[22] mapped the resistance gene xa34（t） to pathotype V in BG1222. Both xa5 and xa34（t） are recessive resistance genes， suitable for conventional rice breeding.

Utilization of Resistance Gene xa5 Effective to the Strong Virulence Pathotype V of Rice Bacterial Blight

New disease-resistant varieties bred with IRBB5 carrying gene xa5

Our research group have successively bred new rice varieties Baixiangzhan， Baijingzhan and Baisizhan using IRBB5， an international rice variety containing the recessive gene xa5， and high-quality varieties （lines， intermediate materials） resistant to rice blast and susceptible to bacterial blight through hybridization， multiple cross， pedigree selection and synchronous resistance identification of various generations. These three varieties showed resistance to the strong virulence pathotype of bacterial blight and rice blast， good rice quality （level 3 of rice quality of Guangdong）， and production equivalent to or slightly higher than that of the regional test control varieties， reaching the level of the major cultivars in Guangdong. Meanwhile， they exhibited strong resistance to stress and are suitable for production and utilization in areas where bacterial blight exists throughout the year in Guangdong， especially in coastal areas where bacterial blight is severe.

Baixiangzhan is bred based on IRBB5 （xa5） as the source of resistance and high-quality susceptible varieties Qijingzao 2-4， Qisizao 21 and Jiuqixiangzhan in South China through hybridization and multiple cross （IRBB5/Qijingzao 23-4）//Qisizao 21///Jiuqixiangzhan）[13]. This variety is the first variety containing a new type of disease resistance gene successfully bred using the international rice gene xa5 resistant to pathyotype V. It is also the first varieties resistant to the strong virulence pathotype V of bacterial blight used in Guangdong and even southern China. The breeding of Baixiangzhan is a major breakthrough in the breeding for resistance to strong virulence bacteria of bacterial blight in rice， which has a far-reaching impact on improving the level of disease resistance breeding in South China and solving the need for the production in coastal areas with the strong virulence pathotype V of bacterial blight. This variety inherits the rice blast resistance of Jiuqixiangzhan， and can also be used as an excellent variety resource resistant to rice blast in South China for rice blast resistance breeding and production in rice blast areas. The genetic map of Baixiangzhan is shown in Fig. 1.

On the basis of the breakthrough of Baixiangzhan resistance， we used Kangbai Jingxianzhan carrying gene xa5 resistant to the pathotype V and the intermediate breeding material with high yield and resistance to rice blast to further select excellent disease-resistant varieties. A new resistant variety， Baijingzhan， was bred using the intermediate material of crossing Wubaili and Jingxian 89 （the female parent has large ears and good quality） and Kangbai Jingxianzhan （the male parent carries the xa5 gene and has better resistance） （Wubaili/Jingxian 89//Kangbai Jingxianzhan） by the pedigree method. This variety maintained the resistance of Baixiangzhan and increased its yield. In 2013， it participated in the regional test of early rice in Guangdong Province， and showed an average yield of 6 495.15 kg/hm2， which increased by 1.18% compared with the control variety 95 Yuxiangyouzhan; and in 2014， it participated in the production test of early rice in Guangdong Province， and showed an average yield of 6 598.65 kg/hm2， which was 5.05% higher than the control variety 95 Yuxiangyouzhan. The daily yield was 46.65-51.60 kg.

Baisizhan with a higher yield was selected with Baixiangzhan resistant to bacterial blight and rice blast as the male parent and high-quality and high-yielding local line Fanfengsimiao in Guangdong Province as the female parent. It was approved by the Guangdong Crop Variety Approval Committee in 2017. The yield performance of Baisizhan： Baisizhan participated in the regional trial of late rice in Guangdong Province in 2016， and showed an average yield of 6 700.80 kg/hm2， which was 0.74% higher than the control variety Yuejingsimiao 2; and it participated in the provincial production test in 2016， and exhibited an average yield of 6 820.95 kg/hm2， which was 1.16% higher than the control variety Yuejingsimiao 2. The daily yield was 56.85-60.60 kg. The growth period of Baisizhan was equivalent to that of the control variety Yuejingsimiao 2. The rice quality was identified as level 3 of rice quality of Guangdong. It was resistant to rice blast and bacterial blight， and moderate in cold tolerance. It is suitable for early and late planting in rice growing areas except the northern part in Guangdong. The main agronomic indicators of Baixiangzhan， Baijingzhan and Baisizhan are shown in Table 3.

In addition， we have also bred two new lines A114 and A738 carrying the recessive gene xa-5， with superior appearance and high yield quality. The restorer line R5 with the recessive gene xa5 will serve as an important bridge parent in conventional high-quality rice and three-line hybrid rice.

Demonstration and production performance of disease-resistant varieties

In recent years， disease-resistant varieties such as Baixiangzhan have been planted in rice areas with the occurrence of the strong virulence pathotype V of bacterial blight， and showed obvious advantages in preventing and controlling bacterial blight. Yangxi County， Guangdong Province is located on the coast of western Guangdong， where typhoons occur frequently. In this area， the late crop of rice production （the second crop of double cropping） is in the typhoon season， and the seedlings often suffer from bacterial blight due to mechanical damage caused by typhoons and rains and floods， so Yangxi County is the area most severely affected by bacterial blight. In late 2014， we set up a rice bacterial blight prevention and control demonstration area in Baishi Village， Shangyang Town， Yangxi County. The disease-resistant varieties Baixiangzhan （resistant）， Xinhuangzhan （resistant）， Baigengzhan （resistant）， Zhenfengyou 9822 （moderately resistant） and Meiyou 9822 （moderately resistant） were used as the main measures to carry out comprehensive prevention and control demonstration of rice bacterial blight， in combination with agricultural cultivation and chemical control methods， and significant results were achieved （Table 4）.

The demonstration results showed that the disease-resistant varieties Baixiangzhan， Xinhuangzhan and Baijingzhan were not treated with any agents， and bacterial blight occurred slightly （incidence rate 0.8%-1.2%， disease index 0.1-0.4）， while the control cultivar was severely diseased （incidence rate 55.6%， disease index 36.2）. The incidence rate of the disease-resistant varieties was reduced by 54.4% to 54.8%， and the control efficiency reached over 99.4%. Compared with the control， the disease occurrence on the disease-resistant varieties was delayed by 12 d. The rice yield increased by 30.16%-47.62%. The control effect of the disease-resistant varieties was also more obvious than other control measures.

Problems and Suggestions

In the breeding of disease-resistant hybrid rice， it is important to select the parents that carry resistance gene resources. However， the resistant parents are usually relatively primitive germplasm with poor production characteristics. For example， IRBB5 carrying the recessive gene xa5 resistant to the strong virulence pathotype V of bacterial blight has small ears and sparse grains， and the economic characteristics are poor. To breed varieties suitable for large-scale production， continuous improvement is needed. We first transformed IRBB5 into a high-quality line carrying the xa5 recessive resistance gene， Kangbaizhan. The economic traits of Kangbaizhan have also been improved， which is a big improvement， but it is not resistant to rice blast. Kangbaizhan was then bred into a double-resistant high-quality variety Baixiangzhan， which is also resistant to rice blast. The variety has multiple resistance and broadened use and application value. However， Baixiangzhans high-yielding performance needs to be improved. On the basis of combining the high quality and double resistance of Baixiangzhan， it was combined with good-quality high-yield local lines in Guangdong， producing Baigengzhan and Baisizhan， which maintain the strong resistance and good quality of Baixiangzhan and have better yielding ability.

In conventional rice breeding， recessive genes are easier to utilize than dominant genes. After the progeny of hybrid F2 is artificially inoculated with the strong virulence pathotype of bacterial blight， a stable disease-resistant line can be selected， and combined with high-yield traits， rice quality and identification of resistance to rice blast， the realization of comprehensive breeding can be speeded up. The successful breeding of Baixiangzhan， Baijingzhan and Baisizhan and their large-scale production and utilization are examples.

The inheritance of the resistance to the strong virulence pathotype V of rice bacterial blight determines that the disease resistance of plants is a potential of the organism itself. The resistance to rice bacterial blight is mainly divided into horizontal resistance and vertical resistance. The horizontal resistance is co-controlled by minor polygenes， that is， the accumulation of multiple minor genes. This type of resistance is relatively stable and not easy to lose， which is conducive to the breeding of durable resistant varieties. The vertical resistance is generally controlled by 1-2 pairs of major resistance genes， that is， controlled by major genes. This type of disease resistance genes generally shows strong resistance to specific races， and resistance to one or several specific pathotypes of bacterial blight. In the breeding for resistance to the strong virulence pathotype V of rice bacterial blight， IRBB5 （xa5）， BG1222[Xa34（t）] and CBB23 （Xa23） are known as non-allelic disease resistance genes. Through hybrid breeding and molecular assisted markers， two or more resistance genes can be effectively aggregated， and combined with rice blast resistance， new rice varieties carrying multiple resistance genes can be breed， so as to improve the durable resistance of the varieties.

xa5 is a recessive gene， and varieties with stable resistance can be obtained quickly after hybridization due to rapid acquisition of homozygosis for the resistance gene， so it is an excellent gene for conventional rice breeding. However， the recessive gene has limitations in its direct application in hybrid rice due to non-expression of resistance in F1. Researcher Zhang et al. from the Institute of Crop Sciences， Chinese Academy of Agricultural Sciences[23-25] identified a new bacterial blight resistance gene Xa23 from common wild rice， which is a fully dominant resistance gene throughout the growth period with a wide resistance spectrum and strong resistance introduction effect. It can be used in hybrid rice breeding for disease resistance.

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