Yusheng LI， Juan YANG, Shengdeng HUANG

Institute of Food Crops, Jiangsu Academy of Agricultural Sciences; Jiangsu High Quality Rice Research and Development Center,Nanjing 210014, China

Rice false smut is a kind of common fungal disease,which is a panicle disease caused by Ustilaginoidea virens (Cooke.) Takah.Rice false smut is widely distributed all over the major rice areas. Among them, rice false smutis the heaviest in Asia. Japan, China and Philippines have the most harmful diseases[1].Rice false smut occurs each year in the growth region of japonica rice in China, and has the characteristics of heavy incidence of the disease, wide range, high frequency, great loss and so on. Rice false smut has risen from the original secondary disease to the main disease at present, which is one of the important diseases seriously threaten rice. Since the 1980s, rice false smut becomes more serious and affects the quality and yield of rice with the extension of hybrid rice, the rapid promotion of high-yield varieties, and the increasing nitrogen application[2-6].Besides, false smut balls contain harmful toxin to both human and animal. Long-term consumption can significantly restrict the tubulin of animal[7-9]. Previous researches have shown that using rice with false smut as the feed for mice can cause tissue necrosis of liver, kidney, bladder and so on[2,10].

In-depth research has been carried out on the control and occurrence regularity of rice false smut. Bacteri-cide and regulating sowing time are usually used to avoid the disease epidemic period, so as to control the occurrence and spread of rice false smut[11-13]. However, these methods can not fundamentally prevent rice false smut; and large amount of bactericide use usually causes environmental pollution.At present,researches on the resistant mechanisms and disease resistance genetic research of rice false smut is still at its initial stage;and the research results have not been applied in practical production.Li et al.found out that rice false smut was controlled by two pairs of equivalent host genes + minor-polygene based on host gene+polygene mixed inheritance model[14]. Host disease resistance genes was genetically stable under different environments in different years[15-16].Xu Jian-long et al.detected two QTLs related to disease resistance by graphic genotypes overlap method[17].

Lagging of research on disease resistance genetic mechanism and disease resistance genes of rice false smut seriously restricted the development of rice breeding for disease resistance. Based on theses, molecular markers tightly linked to resistance genes were found out by locating new resistance genes, especially the disease resistance genes of a single pathogenic strain. Disease-resistant variety was selected by molecular marker assisted method; and new breeding way for rice false smut was found.

Materials and Methods

Materials

Daguandao was a local japonica rice variety in Tai Lake rice area. IR28 is an indica rice variety from Philippines-based International Rice Research Institute. Test materials for evaluation of disease resistance were 157 recombinant inbred lines (RILs)derived from an inter-subspecies cross of Daguandao/IR28. Artificial inoculation identification showed that Daguandao was susceptible to disease, while IR28 was resistance to disease. Molecular genetic map for analysis included 167 SSR molecular markers, which were evenly distributed in 12 chromosomes.The molecular mapping was 1846.6 cM in length and the average distance between markers was 11.1 cM.

Bacterium for inoculation was the hyphae-spore mixture of single pathogenic bacteria strain Pi-1. Spore concentration was 100-150 spores/vision under microscope with 100 times.

Methods

Material planting Two parents and 157 RILs caused by the strain Pi-1 of rice false smut were planted in special plants pool for disease identification in Nanjing City. Each RIL had 15 plants with two repetitions. The planting spacing was 12 cm 15 cm. Single seedling was planted; bactericide was not used during the whole growth period; and conventional cultivation management was adopted.

Pi-1 inoculation Artificial inoculation method of rice false smut was adopted[18-19]. One week before boot rupturing stage,inoculation fluid was injected to the side of ear buds by an injector on16:00. The inoculation amount was 1-2 ml/ear. A total of 10 plants were inoculated, with 3 ears for each plant.Besides, 5 rice plants were taken as the control. After inoculation, moisturizing spray was carried out according to the climate changes.

Evaluation of disease resistance

Three weeks after inoculation,lines without diseases resistance showed disease symptom. Incidence was investigated from this time; the diseased grain number on each ear was counted. With diseased grain number on ear with the most serious illness as the index, grading was carried out according to the standard of Tang Chun-sheng et al[20-21]. Grade 0 indicated no disease;grades Ⅰand Ⅱindicated one and two diseased grains on each ear;grade Ⅲand Ⅳwere 3-5 and 6-9 diseased grains on each ear; grade Ⅴwas more than 10 diseased grains on each ear. The diseased plant number and disease grade of two parents and RILs were obtained.Disease index was calculated by disease index equation; the result was converted to phenotypic value after arcsine transformation, so as to calculate QTL.

QTL analysis

QTL Cartographer2.5 software was adopted.QTL positioning and genetic parameter estimates were carried out by composite interval mapping. Test threshold LOD=2.5 was obtained[22]. QTL naming was carried out by the method of Mc Couch in 1997[23].

Results and Analysis

Resistance reaction of two parents to rice false smut

Parent IR28 had no or very slightly disease symptom in two years test,disease indexes of which were 0 and 0.002, respectively. According to resistance grade, the IR28 belonged to high resistance to rice false smut.Disease indexes of Daguandao in the two years were 76.3 and 79.4, which was highly susceptible(Table 1),

Resistance reaction of RIL population to rice false smut

In the two years, 157 RILs showed continuous distribution in both HR and HS. Population disease indexes were distributed in the ranges of 0.0-80.7 and 0.0-83.4,respectively(Fig.1). Distribution of population disease indexes showed continuous distribution in highly resistant,moderately resistant, moderately susceptible and highly susceptible lines, indicating that resistance of this population to rice false smut was controlled by host gene and minor modification genes.

Table 1 Reaction of parents to rice false smut by artificial inoculation at booting stage

Rice false smut QTL detection and effects analysis

In 2012, four QTLs (qFsr2a, qFsr2b, qFsr8a, qFsr11)in Pi-1 strain of rice false smut were detected. Their LOD values were 5.8,3.6,3.2 and 9.6,respectively.The contribution rate was between 8.7% and 16.9% ; additiveeffect was within -9.9-7.6 (Table 2).The four QTLs were located on chromosomes 2,8 and 11 (Fig.2).qFsr2a,qFsr8a and qFsr11 were allelic genes from parent IR28; the additive values were -8.7, -6.7 and -9.9, respectively.The effects of actions reduced the disease index of population, the range of which was from -9.9 to -6.7. However, the rice resistance to rice false smut enhanced. As for qFsr2b from parent Daguandao,additive value was positive; disease index enhanced; effects of actions reduced the rice resistance to rice false smut.

In 2013, four QTLs (qFsr7, qFsr8b, qFsr11, qFsr12) were detected,which were located on chromosomes 7,8, 11 and 12 (Fig.2). Among them,additive value of qFsr7 was 7.1, which was the same as the qFsr2b detected in 2012. Both qFsr7 and qFsr2b were from the allelic genes of parent Daguandao. qFsr8b, qFsr11 and qFsr12 were all from the allelic genes of parent IR28. Their contribution rates were 8.5%-17.2%; and additive values were between -14.3 and -6.5(Table 2).

In the two years, seven QTLs of rice false smut were detected. Among them, qFsr11 was detected in both 2012 and 2013. In the RM26281 -RM3701 interval of chromosome 11,distance of molecular marker RM3701 was 0.16 cM. This locus had relatively high genetic stability in two years. The additive values of qFsr11 were -9.9 and -14.3 in 2012 and 2013, respectively. And the contribution rates were 13.5%and 17.2%(Table 2).

Table 2 Chromosome location and characteristics of QTLs for rice false smut resistance

Discussions

Occurrence of rice false smut was easily affected by the environment,especially that the changes of temperature and humidity had strong regulatory effects on the infection and spread of rice false smut. Therefore, it wasvery important to successfully induce the rice false smut and to select scientific method of resistance identification. In this research, artificial injection vaccination was adopted; the inoculation solution was hyphae-spore mixture liquid, which ensured the thoroughly exposed to Pi-1 strain and the fully infection. Zhang Jun-cheng et al.found out that this method had strong induced ability and good incidence efficiency[18].The maximum incidence rate reached 100% and disease index was 85.5. Therefore, this method had better effects than single conidium inoculation, which had only 11%-16% incidence rate[24]. At the same time, this method was more scientific and reliable than spray inoculation method[25],bacterium liquid coating inoculation method[12], natural infection in open field[26]and so on. In this research, the incidence rate of infected lines reached 90.6%;while the control showed no disease signs, showing that the disease was caused by inoculation.

There were anti-disease genes in disease-resistant variety, which was determined by genetic characteristics in variety itself. Few anti-disease genes were detected in susceptible varieties. Kunihiro Y[27]et al. found out that Jingxi 17 had resistance genes to sheath blight. Ding Xiu-lan et al. detected resistance genes to stripe leaf blight in Kinmaze. In this research,Daguandao was susceptible parent;the alleles resistance genes qFsr2b and qFsr7 were detected in its offspring. Thus, it could be concluded that due to the gene interaction in pure line cultivar, a few resistance genes could not be expressed. After genetic recombination, gene combination changed so that resistance of genes was expressed. The existence mode of this resistance gene would be a new research subject.

In this research, qFsr11 was detected in both 2012 and 201 on chromosome 11, showing that this locus had high genetic stability and could stably enhance the resistance of rice to diseases. Resistance stability of mixed strain of rice false smut was researched under different environments in different years. Results showed that there was genetic stability region in terminal chromosome 11 had,which had several disease-resistant QTLs[15-16]. The incidence rate and disease index of single pathogenic strain were lower than those of mixed strains, because of the cumulative effects of pathogenicity of various mixed strains, which formed relatively strong selection pressure and strengthened the effects of disease.

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