Evaluation and Identification of Resistance to Diseases and Pests for Rice Varieties in Hainan

Qingjie TANG Xinjie CEN Liqiong TANG Jianxiao CHEN Xiaowei YAN

Abstract [Objectives] This study was conducted to select multi-resistant rice varieties in Hainan.

[Methods] Seven new rice germplasms were identified by naturally induced disease nursery， field spraying and artificial inoculation.

[Results] The restorer line Haihui 1558 was resistant to the dominant races ZB and ZC of rice blast and Tryporyza incertulas， and moderately resistant to pathotype IV of bacterial blight. The restorer line Haihui 227 was resistant to pathotype IV of bacterial blight and T. incertulas. Red rice Hainonghong 1 was resistant to pathotype IV of bacterial blight， and moderately resistant to the dominant races ZB and ZC of rice blast. Black glutinous rice Haifengheinuo 2 was moderately resistant to pathotypes IV of bacterial blight and T. incertulas.

[Conclusions] The study provides reference for utilization of rice breeding on resistance to diseases and pests.

Key words Rice landrace; Rice blast; Bacterial blight; Tryporyza incertulas; Brown planthopper; Identification

White-backed planthoppers and brown planthoppers are the main pests in East Asia， South Asia， Southeast Asia and other places[1]. The occurrence area of rice planthoppers in Hainan from 2010 to 2011 was close to 2.667 × 105 hm2. Bacterial blight is an important bacterial disease on rice. The disease frequently breaks out in southern China， central and eastern China， and other Southeast Asian rice regions， and is one of the important limiting factors for high and stable rice yield[2-3]. In the late 1980s， a strongly-virulent pathotype V of rice bacterial blight appeared in Guangdong Province. None of the varieties planted in production at that time could resist the pathogenic type. The pathotype V bacteria exacerbated the development of bacterial blight in Guangdong Province and spread to other provinces， posing a great threat to rice production， so there was an urgent need for disease resistant varieties[4]. Rice blast is also an important disease in rice production in South China. In recent years， it has become more and more serious. Breeding and using resistant varieties is the most economical， effective and safe measure to control rice blast[5].

At present， there have been some reports on rice blast and bacterial blight in Hainan[6]， but the main focus is on the isolation and pathogenicity analysis of the dominant physiological races of rice bacterial blight. The identification of resistance to such four major pests and diseases as rice blast， bacterial blight， rice planthoppers and Tryporyza incertulas of Hainan Province has not been conducted. In this study， in order to select disease-resistant and pest-resistant varieties （combinations） suitable for use in this region and improve the rice production level in Hainan Province， resistance identification by field spraying and inoculation were carried out on 7 new rice germplasms of different types， with a view to providing reference for breeding of rice varieties resistant to diseases and pests in Hainan Province.

Materials and Methods

Identification of rice blast

Tested varieties and inoculated strains

Tested varieties： Shanlannuo 2; Shanlanlu 1; Huaxiang; Hainonghong 1; Haifengheinuo 2; Haihui 1558; Haihui 227 （provided by the Cereal Crops Institute， Hainan Academy of Agricultural Sciences）; Guangluai 4 （susceptible control variety， provided by the Institute of Plant Protection， Guangdong Academy of Agricultural Sciences）.

Artificial inoculated strains： Dominant races ZB and ZC of rice blast in southern China （provided by the Institute of Plant Protection， Guangdong Academy of Agricultural Sciences）.

Identification method of rice blast

Identification by artificial inoculation at seedling stage： The representative physiological races in recent years were selected. The seedlings were spray-inoculated at the 3- to 4-leaf stage. The temperature during inoculation should be 25-28 ℃. After inoculation， the seedlings were placed under a constant temperature of 25-28 ℃ and covered to keep wet in dark for 24 h， and then sprayed with water on time to keep wet. After 7-10 d， when the susceptible control variety was diseased fully， the leaf blast grade was investigated[7].

Identification of neck blast by naturally induction in the field： In the test area， the rice areas with frequent fog and dew condensation for a long time where the disease often occurs were selected. The fields with fertile soil and convenient irrigation and drainage were selected. After soaking seeds and germinating the tested varieties， sowing in strip or transplanting were adopted.  Susceptible varieties were planted around the tested varieties. Nitrogen fertilizer was applied once before heading， which was beneficial to break of the disease. The condition of neck blast was investigated after yellow ripe[7].

Disease division and evaluation criteria： The survey criteria for leaf blast and neck blast referred to the identification criteria for disease resistance in national regional tests. The resistance evaluation was carried out according to resistance index， and the resistance index of each variety was calculated according to Resistance index=Average disease grade of leaf blast×25%+ Disease grade of neck blast incidence×25%+ Disease grade of neck blast loss index×50%[7].

Identification of bacterial blight

Tested varieties and inoculated strains

The tested varieties included Shanlannuo 2; Shanlanlu 1; Huaxiang; Hainonghong 1; Haifengheinuo 2; Haihui 1558; Haihui 227 （provided by the Cereal Crops Institute， Hainan Academy of Agricultural Sciences）; Jingang 30 （susceptible control variety， provided by the Institute of Plant Protection， Guangdong Academy of Agricultural Sciences）.

The representative dominant pathotype IV of bacterial blast was selected. The bacterial strain was cultivated on Coburns medium slant for 48-72 h. The bacterial liquid was washed with sterile water to prepare 300 million bacteria/ml bacterial liquid[7] （Provided by the Institute of Plant Protection， Guangdong Academy of Agricultural Sciences）.

Identification method of bacterial blight

Identification by artificial inoculation method： The seedling age of late rice was controlled within 25 d; each variety was planted in one row with 10 holes in sequence， each of which was planted with one seedling， according to a row spacing of 17 cm × 23 cm， and the susceptible variety was planted every 10 varieties as control; the fertilization level of the identification nursery was higher than that of the general field; and inoculation was performed by cutting leaves at the fully tillering stage of rice. At dusk， the top of the leaves of one row of plants was cut 2 cm long with surgical scissors dipped in the bacterial liquid， and 5 leaves was inoculated per hole[7]. 20 d after the inoculation， the disease conditions were investigated when the disease of the susceptible control variety reached grade 9 in accordance with Descriptors and Data Standard for Rice Germplasm Resources[8].

Identification of resistance to T. incertulas

Tested varieties and T. incertula population

The tested varieties included Shanlannuo 2， Shanlanlu 1， Huaxiang， Hainonghong 1， Haifengheinuo 2， Haihui 1558， Haihui 227 （provided by the Cereal Crops Institute， Hainan Academy of Agricultural Sciences）， TKM6 （pest-resistant control variety） and TN1 （susceptible control variety， preserved in the laboratory of the Institute of Plant Protection， Guangdong Academy of Agricultural Sciences）. The egg mass was collected from the field in August 2016. After the larvae were hatched， they were attached to the seedlings.

Testing method

Seedling identification： The rice varieties to be tested were sown in 50 cm × 40 cm and 25 cm deep plastic pots， 20-30 seeds per variety， with 3 repetitions， randomly arranged， and the susceptible control variety TN1 and resistant control variety TKM6 were also planted. At 10 d after seed germination， 10-15 strong seedlings were kept in each treatment. When the seedlings grew to 3-4 tillers， the first-instar larvae of T. incertulas were inoculated with 10 heads per hill. Seven days after the inoculation， the number of seedlings with dead heart was investigated， and following formulas （1） and （2） were used to calculate the dead heart rate and corrected dead heart rate. The test is effective under the condition that the dead heart rate reaches 25%.

（1）： Dead heart rate=Number of plants with dead heart/Total number of seedlings×100%

（2）： Correlated dead heart rate=（Dead heart rate of tested variety/Dead heart rate of control susceptible to pests）×100%

Identification of resistance to rice planthoppers

Tested varieties and tested brown planthopper population

The tested varieties included Shanlannuo 2， Shanlanlu 1， Huaxiang， Hainonghong 1， Haifengheinuo 2， Haihui 1558， Haihui 227 （provided by the Cereal Crops Institute， Hainan Academy of Agricultural Sciences）， RATHU HEENATI （pest-resistant control variety） and TN1 （susceptible control variety） （preserved in the laboratory of the Institute of Plant Protection， Guangdong Academy of Agricultural Sciences）. The brown planthopper population to be tested was collected from the field and raised in the laboratory for 6 generations， which were biotype 1 and 2 mixed populations.

Detection method

Seedling identification： The rice varieties to be tested were sown in 40 cm×30 cm and 10 cm deep plastic pots， 30-40 seeds per variety， with 3 repetitions， randomly arranged， and the susceptible control variety TN1 and resistant control variety RATHU HEENATI were also planted. The seedlings were thinned on the 4th day after seed germination， and 30 strong seedlings were kept in each row. When the seedlings grew to 3 leaves， 2- to 3-instar brown planthopper nymphs were input according to an average of 5 pests per seedling. When the pest damage in the susceptible control variety TN1 reached grade 9， grading was performed. The obtained data was evaluated by weighted average.

Results and Discussion

Identification results of rice blast resistance

The identification results of resistance in the 7 germplasms to rice blast were of following five types： highly resistant， resistant， moderately resistant， susceptible， and highly susceptible. Haihui 1558 was resistant， Hainonghong 1 was moderately resistant， and other germplasms were moderately susceptible， susceptible and highly susceptible.

Identification results of resistance to bacterial blight

The resistance to the dominant pathotype IV of bacterial blight was evaluated and included such three types as resistant， moderately resistant and highly susceptible. The resistant germplasms were Hainonghong 1， Huaxiang and Haihui 227， the moderately resistant germplasms were Haihui 1558 and Haifengheinuo 2， and other germplasms showed high susceptibility （Table 4）.

Identification results of resistance to T. incertulas

The test was repeated 3 times， and SPPSS19.0 software was used for data analysis. The results were expressed as "mean ± standard deviation". When the dead heart rate of the susceptible control variety TN1 reached 100%， and that of pest-resistant control variety TKM6 was 33.07%， the identification was effective. From Table 5， it could be known that the test materials showed different resistance. The varieties exhibiting resistance to T. incertulas were the restorer lines Haihui 1558 and Haihui 227， with a corrected dead heart rate of 21%-40%. The resistant variety wasHaifengheinuo 2 with a corrected dead heart rate of 41%-60%. Huaxiang was susceptible with a corrected dead heart rate of 61%-80%. The varieties with high susceptibility were Hainonghong 1， Shanlannuo 1 and Shanlanlu 1， with a corrected dead heart rate of 100%.

Identification results of resistance to rice planthoppers

When the damage in the susceptible control variety TN1 reached grade 9 and the pest-resistant control variety RATHU HEENATI averaged grade 3， the tested materials showed different resistance. According to Table 6， Shanlannuo seedlings suffered from damage of grade 5-9， mainly grade 9， and its overall resistance was evaluated to be highly susceptible. The damage of Shanlanlu was grade 9， and its overall resistance was evaluated as highly susceptible. The damage grades of Huaxiang were 5 and 9 and the overall resistance was also evaluated to be highly susceptible. The damage grade of Hainonghong 1 was distributed at 5 and 9 and the overall resistance was highly susceptible as well. The damage in Haifengheinuo 2 was graded at 5 and 9， mainly grade 5， and the overall resistance was susceptible. Haihui 1558 showed damage grades of 5-9， mainly grade 5， and the overall resistance was also evaluated to be susceptible. The damage grade of Haihui 227 was distributed at grades 5 and 9， mostly grade 5， and the overall resistance was evaluated to be susceptible as well.

Conclusions and Discussion

Although there were fewer new rice germplasms available for testing， the tested rice germplasms are of different types， including 2 restorer lines， namely Haihui 1558 and Haihui 227， 3 special rice types， namely fragrant rice Huaxiang and Red rice Hainonghong 1， black glutinous rice Haifengheinuo 2， 2 Shanlan varieties special for Hainan， namely Shanlannuo 2， Shanlanlu 1 and Huaxiang， and Huaxiang.

According to the identification results of the seven new rice germplasms tested， there was one germplasm resistant to rice blast and one germplasm moderately resistant to rice blast. There were 3 germplasms resistant to the dominant pathotype IV of bacterial blight and 2 germplasms moderately resistant to the pathotype IV. There were 2 germplasms resistant to T. incertulas， both of which are restorer lines， and one germplasm moderately resistant to T. incertulas. No materials resistant to rice planthoppers were found.

Although there were few materials for testing， there were germplasms with double resistance and even triple resistance. The restorer line Haihui 1558 was resistant to rice blast and T. incertulas， and moderately resistant to bacterial blight. The restorer line Haihui 227 was resistant to bacterial blight and T. incertulas. Red rice Hainonghong 1 was resistant to bacterial blight and moderately resistant to rice blast. Black glutinous rice Haifengheinuo 2 was moderately resistant to bacterial blight and T. incertulas. It is speculated that the promotion of these new germplasms or varieties in the Hainan rice area can effectively resist the occurrence of pests and diseases.

References

[1] GONG SL， HOU ML. Research progress on rice varietal resistance to the brown planthopper and white-backed planthopper[J]. Plant Protection， 2017， 43（1）： 15-23. （in Chinese）

[2] YANG J， WANG HF， LIN F， et al. Analysis of different rice varieties （lines） resistance to bacterial leaf blight[J]. Southwest China Journal of Agricultural Sciences， 2017， 30（9）： 1991-1996. （in Chinese）

[3] WANG MM， ZHANG F， SHI YY， et al. Evaluation of bacterial blight resistance of Chinese micro core rice germplasm[J]. Journal of Plant Genetic Resources， 2017， 18（4）： 771-777. （in Chinese）

[4] CHENG TH， WANG CY， CHEN S， et al. Breeding and application of new rice variety Baisizhan resistant to strong-virulence pathotype V of bacterial blight[J]. China Seed Industry， 2017（12）： 63-64. （in Chinese）

[5] WANG WJ， ZHOU JY， WANG CY， et al. Distribution of eight rice blast resistance genes in indica hybrid rice in China[J]. Chinese Journal of Rice Science， 2017， 31（3）： 299-306. （in Chinese）

[6] XIA LQ， LI MR， XIE SM， et al. Isolation and pathogenicity analysis of the predominant race of Xanthomonas oryzae pv. oryzae in Hainan[J]. Molecular Plant Breeding， 2016， 14（5）： 1336-1340. （in Chinese）

[7] ZHU XY， ZENG LX， GAO HL， et al. Identification and screening of disease resistance of new indica rice varieties in South China[J]. Guangdong Agricultural Sciences， 2010， 37（9）：12-15. （in Chinese）

[8] HAN LZ， WEI XH， CAO GL， et al. Descriptors and data standard for rice germplasm resources[M]. Beijing： China Agriculture Press， 2006：112-113，116-118. （in Chinese）