Gapless Genome Assembly of ZH8015 and Preliminary Multi-Omics Analysis to Investigate ZH8015's Responses Against Brown Planthopper Infestation

Accurate genomic information is essential for advancing genetic breeding research in specific rice varieties.This study presented a gapless genome assembly of the indica rice cultivar Zhonghui 8015(ZH8015)using Pac Bio HiFi,Hi-C,and ONT(Oxford Nanopore Technologies)ultra-long sequencing technologies,annotating 43037 gene structures.Subsequently,utilizing this genome along with transcriptomic and metabolomic techniques,we explored ZH8015's response to brown planthopper(BPH)infestation.Continuous transcriptomic sampling indicated significant changes in gene expression levels around 48 h after BPH feeding.Enrichment analysis revealed particularly significant alterations in genes related to reactive oxygen species scavenging and cell wall formation.Metabolomic results demonstrated marked increases in levels of several monosaccharides,which are components of the cell wall and dramatic changes in flavonoid contents.Omics association analysis identified differentially expressed genes associated with key metabolites,shedding light on ZH8015's response to BPH infestation.In summary,this study constructed a reliable genome sequence resource for ZH8015,and the preliminary multi-omics results will guide future insect-resistant breeding research.

Synergistic effects of the entomopathogenic fungus Isaria javanica and low doses of dinotefuran on the efficient control of the rice pest Sogatella furcifera

The rice planthopper,Sogatella furcifera,is a piercing-sucking insect pest of rice,Oryza sativa.It is responsible for significant crop yield losses,and has developed moderate to high resistance to several commonly used chemical insecticides.We investigated the effects of the insect fungal pathogen Isaria javanica,alone and in combination with the chemical insecticide dinotefuran,on S.furcifera under both laboratory and field conditions.Our results show that I.javanica displays high infection efficiency and mortality for different stages of S.furcifera,reducing adult survival,female oviposition and ovary development.Laboratory bioassays showed that the combined use of I.javanica with a low dose(4-16 mg L^(-1))of dinotefuran resulted in higher mortality in S.furcifera than the use of I.javanica or dinotefuran alone.The combined treatment also had more significant effects on several host enzymes,including superoxide dismutase,catalase,peroxidase,and prophenol oxidase activities.In field trials,I.javanica effectively suppressed populations of rice planthoppers to low levels(22-64%of the level in untreated plots).Additional field experiments showed synergistic effects,i.e.,enhanced efficiency,for the control of S.furcifera populations using the combination of a low dose of I.javanica(1×10^(4) conidia mL^(-1))and a low dose of dinotefuran(~4.8-19.2%of normal field use levels),with control effects of>90%and a population level under 50 insects per 100 hills at 3-14 days post-treatment.Our findings indicate that the entomogenous fungus I.javanica offers an attractive biological control addition as part of the integrated pest management(IPM)practices for the control of rice plant pests.

Analysis of Resistance-related Proteins in Rice Against Brown Planthopper by Two-dimensional Electrophoresis

A recombinant inbred population (RI) was constructed from a cross between B5, an introgression. line from the wild rice Oryza officinalis Wall. ex Watt, and susceptible cultivar Minghui 63 ( O. sativa L.). The brown planthopper ( BPH) resistances of RI lines were evaluated. Based on bulked segregant analysis (BSA), two protein bulks were made by extracting proteins from equally mixed seedlings of extremely resistant and susceptible plants selected from the RI population, respectively. Two-dimensional electrophoresis was used to detect the changes of polypeptide pattern. Results showed that a protein P40 ( pI 6.3, Mw 40 kD) was significantly reduced or vanished after BPH infestation for 48 h in the susceptible bulk, while it remained uninfluenced in the resistant bulk. In connection with the physiological changes of the resistant and susceptible lines subjected to BPH sucking, we suppose that the protein P40 is related to the interaction responses of lice plants to BPH infestation.

Spectrum Detection of Rice Planthopper Populations from Canopy Reflectance Based on Principal Component Regression

[ Objective] Aiming at problems of early warning for occurrence of rice pests and dynamic monitoring of rice planthopper in field, a detection model for rice planthopper populations was established based on PCR with spectrum detection technology, r Method] Canopy reflectance data were collected using FieldSpeo 3 spectrometer in paddy field, and rice planthoppers populations in hundred hills were detected simultaneously. The sample size was 71, and there were 51 samples in the calibration set and 20 samples in the prediction set. Modeling band was 350 -1 139 nm, and the original spectra were pretreated by first order differential. [ Result] The correlation coefficient of measured values and predictive values was 0. 78, and the RMSEP was 161. [ Conlmion] Spectrum detection was able to be used in investigation and forecasting of rice planthoppere.

Relationship between Rice Planthopper Occurrence Area in China and Atmospheric Circulation Indices

[Objective] This study aimed to establish models based on atmospheric cir- culation indices for forecasting the area attacked by rice planthopper every year, and to provide guide for preventing and controlling planthopper damage. [Method] The data related to rice planthopper occurrence and atmospheric circulation were collected and analyzed with the method of stepwise regression to establish the prediction models. [Result] The factors significantly related to the area attacked by rice plan-thopper were selected. Two types of prediction models were established. One was for Sogatella furcifera （Horvath）, based on Atlantic-Europe circulation pattern W in October in that year, Pacific polar vortex area index in October in that year, North America subtropical high index in August in that year, Atlantic-Europe circulation pattern W in June in that year, northern boundary of North America subtropical high in February in that year, Atlantic-Europe polar vortex intensity index in October in that year and Asia polar vortex intensity index in November in the last year; the other type of prediction models were for Nilaparvata lugens （Stal）, based on the Eastern Pacific subtropical high intensity index in July in that year, northern hemi- sphere polar vortex area index in October in the last year, Asia polar vortex strength index in November in the last year, north boundary of North America-At- lantic subtropical high in September in that year, north boundary of North Africa-At- lantic-North America subtropical high in January in that year, sunspot in September of the last year and eastern Pacific subtropical high area index in September in that year. [Conclusion] With the stepwise regression, the forecasting equations of the rice planthopper occurrence established based on the atmospheric circulation indices could be used for actual forecast.

Study on the Meteorological Condition Grade Forecast for the Migration of Rice Planthopper

By comparative analysis on the meteorological conditions and occurrences of rice planthoppers in Guilin of Guangxi during recent 5 years,the temperature,precipitation,wind direction,wind velocity,humidity were chosen as the factors that affected the migration of rice planthopper in Guilin.Thinking of meteorological conditions and injurious number in early period,regression analysis method was used to establish the work system for grade forecast of meteorological conditions that affected the migration of rice planthopper.The forecast factors and targets were divided as 5 grades in the work system.Using one-week's weather forecast conclusion that local observatory had published,whether the meteorological conditions and injurious number were favorable or not was analyzed synthetically.The meteorological conditions grades that affected the migration of rice planthopper for each day in future 1-7 days were predicted.This is a practical forecast work system and the forecast accuracy for each day is larger than 70%.The work system has positive function in the manufacture practice.

Density Restriction Effects of Reproductive Rate of Rice Brown Planthopper Population in Super Rice Yongyou 6

[ Objective ] The paper was to study the density restriction effects of reproductive rate of rice brown planthopper （BPH） population in super indiea and japonica hybrid rice Yongyou 6. [Method] Different amounts of rice brown planthoppor were released to super indiea and japonica hybrid flee Yongyou 6 during fillering, booting and gain filling stage, respectively. The reproductive rate and variation dynamics of rice brown planthoppor in super rice was also investigated and analyzed. [Result] Tillering stage： the population quantity of rice brown planthoppor during booting and gain filling stage gradually increased with the increasing basal amount of its population during tillering stage; when the population density of rice brown planthopper during grain filling stage reached a certain limit （ 〉 250 head/cluster）, the population density during milking stage showed decrease trend with the increasing of such density; the development of rice brown planthoppor population in super rice Yongyou 6 showed the characteristics of fast in middle and slow in two ends. Booting stage： the development of rice brown planthopper pop- ulation from booting to gain filling stage increased with the increasing basal amount of density, and their correlation during milking stage was not significant; flee brown planthopper population still had high reproduction capacity in the middle and late stage of super rice, which had latent risk on rice yield. Gain filling stage ： when population density of rice brown planthopper 〈 70 head/cluster, the development of it population increased with the increase of basal amount of density, which had great threat to yield. [ Conclusion] The result provided theoretical basis for establishment and improvement of monitoring early warning system, cost-ef- fective control of occurrence and damage of rice brown planthopper, and improvement of the sustained control ability against rice brown planthopper.

Automated Counting of Rice Planthoppers in Paddy Fields Based on Image Processing

A quantitative survey of rice planthoppers in paddy fields is important to assess the population density and make forecasting decisions. Manual rice planthopper survey methods in paddy fields are time-consuming, fatiguing and tedious. This paper describes a handheld device for easily capturing planthopper images on rice stems and an automatic method for counting rice planthoppers based on image processing. The handheld device consists of a digital camera with WiFi, a smartphone and an extrendable pole. The surveyor can use the smartphone to control the camera, which is fixed on the front of the pole by WiFi, and to photograph planthoppers on rice stems. For the counting of planthoppers on rice stems, we adopt three layers of detection that involve the following：（a） the first layer of detection is an AdaBoost classifier based on Haar features;（b） the second layer of detection is a support vector machine（SVM） classifier based on histogram of oriented gradient（HOG） features;（c） the third layer of detection is the threshold judgment of the three features. We use this method to detect and count whiteback planthoppers（Sogatella furcifera） on rice plant images and achieve an 85.2% detection rate and a 9.6% false detection rate. The method is easy, rapid and accurate for the assessment of the population density of rice planthoppers in paddy fields.

The Influence of Transgenic cry1Ab/cry1Ac,cry1C and cry2A Rice on Non-Target Planthoppers and Their Main Predators Under Field Conditions

Transgenic Bt rice has been shown to be an effective means of controlling Lepidoptera pests of rice. However, the potential roles of transgenic rice on planthoppers and their predators need to be investigated before its commercialization. Population density, species dominance and population dynamics are important parameters of arthropods populations in field. So the impacts of three transgenic Bt rice strains expressing crylAb/crylAc, crylC and cry2A on population density, species dominance and population dynamics of three species of planthoppers （Nilaparvata lugens, Sogatella furcifera and Laodelphax striatellus） and their three main predators （ Cyrtorhinus lividipennis, Pirata subpiraticus and Theridium octomaculatum） were evaluated at three sites in Hubei Province, China, in the current study. The results showed that among three species of planthoppers, both in transgenic and non-transgenic rice field, the predominant species ofplanthoppers within phytophagous guild was S. furcifera at any site either growing season （46-50%）. Significantly higher population density ofN. lugens was observed in T2A-1 field relative to Minghui 63 field at Wuxue in 2010. The species dominance of predator, P. subpiraticus, in TT51 field was significantly higher than that in T 1 C-19 and T2A-1 fields in 2009 at Xiaogan site. Sampling date significantly influenced six arthropods except for P. subpiraticus in 2010. The interaction between rice strain^sampling date had no significant adverse effects on the population dynamics of three species of planthoppers and their predators, except for several individual species in 2009. The interaction among rice strain^sampling date^sampling site also had no significant effect on six arthropods except for S. furcifera in 2009. The results indicated that transgenic Bt rice expressing crylAb/crylAc, cry2A and crylC had no significant adverse effects on the population dynamics of three planthoppers and their predators in most investigated data and sampling site.

Induced defense responses in rice plants against small brown planthopper infestation

The small brown planthopper(SBPH), Laodelphax striatellus Fallén(Homoptera: Delphacidae), is a serious pest of rice(Oryza sativa L.) in China. To understand the mechanisms of rice resistance to SBPH, defense response genes and related defense enzymes were examined in resistant and susceptible rice varieties in response to SBPH infestation. The salicylic acid(SA) synthesis-related genes phenylalanine ammonia-lyase(PAL), NPR1, EDS1 and PAD4 were induced rapidly and to a much higher level in the resistant variety Kasalath than in the susceptible cultivar Wuyujing 3 in response to SBPH infestation. The expression level of PAL in the Kasalath rice at 12 h post-infestation(hpi) increased 7.52-fold compared with the un-infested control, and the expression level in Kasalath was 49.63, 87.18, 57.36 and 75.06 times greater than that in Wuyujing 3 at 24, 36, 48 and 72 hpi, respectively. However, the transcriptional levels of the jasmonic acid(JA) synthesis-related genes LOX and AOS2 in resistant Kasalath were significantly lower than in susceptible Wuyujing 3 at 24, 36, 48 and 72 hpi. The activities of the defense enzymes PAL, peroxidase(POD), and polyphenol oxidase(PPO) increased remarkably in Kasalath in response to SBPH infestation, and were closely correlated with the PAL gene transcript level. Our results indicated that the SA signaling pathway was activated in the resistant Kasalath rice variety in response to SBPH infestation and that the gene PAL played a considerable role in the resistance to SBPH.

Pyramiding blast,bacterial blight and brown planthopper resistance genes in rice restorer lines

Rice blast, bacterial blight （BB） and brown planthopper （BPH） are the three main pests of rice. This study investigated pyr-amiding genes resistant to blast, BB and BPH to develop restorer lines. Ten new lines with blast, BB and/or BPH resistance genes were developed using marker-assisted selection （MAS） technique and agronomic trait selection （ATS） method. Only HR13 with resistance genes to blast, BB and BPH was obtained. In addition to blast and BB resistance, four lines （HR39, HR41, HR42, HR43） demonstrated moderate resistance to BPH, but MAS for BPH resistance genes were not conducted in developing these four lines. These data suggested that there were unknown elite BPH resistance genes in the Zhongzu 14 donor parent. A more effective defense was demonstrated in the lines withPi1 andPi2 genes although the weather in 2012 was favorable to disease incidence. Blast resistance of the lines with a single resistance gene,Pita, was easily inlfuenced by the weather. Overal, the information obtained through pyramiding multiple resistance genes on developing the restorer lines is helpful for rice resistance breeding.

Donors for Resistance to Brown Planthopper Nilaparvata lugens(St?l) from Wild Rice Species

Out of 1 989 wild accessions sown in seed boxes for screening, only 1 003 wild accessions with good germination were screened against brown planthopper(BPH), Nilaparvata lugens(St?l) under greenhouse conditions. The collection comprised of accessions from 11 wild species and African cultivated rice. The germplasm was screened for BPH following standard seed box screening technique in the greenhouse. As many as 159 accessions were identified as resistant during the year 2012 based on one year screening. A selected set of BPH resistant accessions were screened again during 2013. Based on the two years screening, seven accessions of O. nivara(AA), one accession of O. officinalis(CC), seven accessions of O. australiensis(EE), five accessions of O. punctata(BB and BBCC) and nine accessions of O. latifolia(CCDD) were confirmed to be resistant to BPH. So far no BPH resistance genes have been identified and designated from O. nivara and O. punctata, hence these may act as new sources of resistance.

LncRNAs are potentially involved in the immune interaction between small brown planthopper and rice stripe virus

Small brown planthopper(SBPH, Laodelphax striatellus Fallén) is an important vector of major crop pathogen rice stripe virus(RSV). Controlling SBPH population is an efficient approach to control RSV. Long non-coding RNAs(lnc RNA) have been reported to block virus replication in hosts. However, the function of lnc RNAs in RSV infection and replication is still unknown. Here, we aimed to study regulatory mechanisms of lnc RNA in an immune system during RSV infection. First, lnc RNA genes were predicted from SBPH transcriptomes using a bioinformatics pipeline based on characteristics of lnc RNA. We identified 4 786 lnc RNA genes corresponding to 5 790 transcripts in SBPH from an RNA-Seq dataset of 15 transcriptomes. Differential expression analysis indicated that 3, 11, and 25 lnc RNA genes were highly expressed in gut, salivary gland, and ovary, respectively, of viruliferous SBPH(Student’s t-test, P<0.05). We randomly selected eight lnc RNAs for expression validation using quantitative real-time PCR, confirming the differential expression of these lnc RNAs between viruliferous and non-viruliferous SBPH. In summary, we present evidence that the expression of lnc RNA genes was induced by RSV infection, suggesting that RSV might be involved in the antivirus immune system in SBPH and participate in regulating the RSV replication mechanism. These data provide helpful information for future investigations of the interaction between lncRNA and RSV.

Expression Analysis of Genes Related to Rice Resistance Against Brown Planthopper, Nilaparvata lugens

Brown planthopper(BPH) is an insect species that feeds on the vascular system of rice plants. To examine the defence mechanism of rice plants against BPH, the pathogenesis-related genes(PR1a, PR2, PR3, PR4, PR6, PR9, PR10a, PR13, PR15 and PRpha), signaling molecule synthesis genes(AOS, AXR, ACO and LOX), antioxidant-related genes(CAT, TRX, GST and SOD) and lignin biosynthesis-related genes(CHS, CHI and C4H) were investigated in a resistant rice variety. AOS, PR6,PR9 and PR15 genes showed significantly increased relative expression levels at 24.38-, 19.17-, 14.71-, and 12.74-fold compared to the control. Moderate increased relative expression levels of lignin biosynthesis-related gene(C4H), pathogenesis-related genes(PR4, PR10a and PRpha), and antioxidant-related gene(GST) were found, while CHI, LOX, SOD, TRX1 and AXR showed decreased relative expression levels. It was thus clearly shown that wound-induced response genes were activated in rice plants after BPH attacks through AOS activation. Jasmonic acid signaling molecule may activate PR6, PR15, GST and CAT subsequently increasing their expression for H_2O_2 detoxification. PR6 were expressed at the highest relative level among the PR genes. These genes therefore have also a considerable synergistic role with the other genes against BPH by interfered their digestion tract system.

Highly Sensitive and Specific Monoclonal Antibody-Based Serological Methods for Rice Ragged Stunt Virus Detection in Rice Plants and Rice Brown Planthopper Vectors

Rice ragged stunt virus（RRSV） is a serious rice disease in Asia, causing serious yield losses on rice. The capsid protein（CP） gene of the major outer capsid protein of RRSV was expressed in Escherichia coli BL21（DE3） using the pMAL-C2 X expression vector. The recombinant protein was used as the immunogen to immunize BALB/c mice. A hybridoma cell line 8A12 secreting monoclonal antibody（MAb） against RRSV was obtained by fusing mouse myeloma cells（Sp 2/0） with spleen cells from the immunized BALB/c mice. Western blot analysis showed that the MAb 8A12 can specifically react with RRSV CP. Using the MAb, an antigen-coated-plate enzyme-linked immunosorbent assay（ACP-ELISA）, a dot enzyme-linked immunosorbent assay（dot-ELISA）, and immunocapture-RT-PCR（IC-RT-PCR） assay were developed to detect RRSV. The established ACP-ELISA, dot-blot ELISA and IC-RT-PCR methods could detect RRSV in infected rice tissue crude extracts with dilutions of 1：40 960, 1：1 280 and 1：655 360（w/v, g mL-1）, respectively. The ACP-ELISA and dot-blot ELISA methods could detect RRSV in infected insect vector crude extracts with dilutions of 1：12 800 and 1：1 600（an individual planthopper μL-1）, respectively. The field survey revealed that Rice ragged stunt disease occurs on rice in Hainan, Yunnan, Guangxi, Sichuan, Guizhou, Fujian, Hunan, Jiangxi and Zhejiang in China.

Ecological Fitness of Non-vector Planthopper Sogatella furcifera on Rice Plants Infected with Rice Black Streaked Dwarf Virus

We evaluated the effects of rice black streak dwarf virus （RBSDV）-infested rice plants on the ecological parameters and its relevant defensive and detoxification enzymes of white-backed planthopper （WBPH） in laboratory for exploring the relationship between RBSDV and the non-vector planthopper. The results showed that nymph survival rate, female adult weight and fecundity, and egg hatchability of WBPH fed on RBSDV-infested rice plants did not markedly differ from those on healthy plants, whereas the female adult longevity and egg duration significantly shortened on diseased plants. Furthermore, significantly higher activities of defensive enzymes （dismutase, catalase and peroxidase） and detoxification enzymes （acetylcholinesterase, carboxylesterase and glutathione S-transferase） were found in WBPH adults fed on infected plants. Results implied that infestation by RBSDV increased the ecological fitness of non-vector planthopper population.

Evaluation of Rice Germplasm for Resistance to the Small Brown Planthopper (Laodelphax striatellus) and Analysis of Resistance Mechanism

One hundred and thirty-eight rice accessions were screened for resistance to the small brown planthopper （SBPH） （Laodelphax striatellus Fallen） by the modified seedbox screening test. Twenty-five rice accessions with different levels of resistance to SBPH were detected, accounting for 18.1% of the total accessions, which included 2 highly resistant, 9 resistant and 14 moderately resistant varieties. Compared with indica rice, japonica rice was more susceptible to SBPH. Antixenosis test, antibiosis test and correlation analysis were performed to elucidate the resistance mechanism. The resistant check Rathu Heenati （RHT）, highly resistant varieties Mudgo and Kasalath, and resistant variety IR36 expressed strong antixenosis and antibiosis against SBPH, indicating the close relationship between resistance level and these two resistance mechanisms in the four rice varieties. Antibiosis was the dominant resistance pattern in the resistant varieties Daorenqiao and Yangmaogu due to their high antibiosis but low antixenosis. Dular, ASD7 and Milyang 23 had relatively strong antixenosis and antibiosis, indicating the two resistance mechanisms were significant in these three varieties. The resistant DV85 expressed relatively high level of antixenosis but low antibiosis, whereas Zhaiyeqing 8 and Guiyigu conferred only moderate antibiosis and antixenosis to SBPH, suggesting tolerance in these three varieties. Antibiosis and antixenosis governed the resistance to SBPH in the moderately resistant accession 9311. Antixenosis was the main resistance type in V20A. Tolerance was considered to be an important resistance mechanism in Minghui 63 and Yangjing 9538 due to their poor antibiosis and antixenosis resistance. The above accessions with strong antibiosis or antixenosis were the ideal materials for the resistance breeding.

Transmission of Rice Black-Streaked Dwarf Virus from Frozen Infected Leaves to Healthy Rice Plants by Small Brown Planthopper (Laodelphax striatellus)

In order to preserve virus for identifying the resistance of rice varieties against rice black-streaked dwarf disease, a simple and reliable method was developed, through which virus-free small brown planthopper （SBPH） acquired rice black-streaked dwarf virus （RBSDV） from frozen infected leaves and the virus was transmitted to healthy rice plants. The experimental results showed that SBPH could obtain RBSDV from frozen infected rice leaves and the virus could be transmitted to a susceptible rice variety. For the ability to acquire RBSDV and transmit the virus to healthy plants by SBPH, there was no significant difference between frozen infected leaves and in vitro infected leaves. The novel method could be applied to identification of rice variety resistance to rice black-streaked dwarf disease, facilitating the breeding process for rice black-streaked dwarf disease resistance.

High Temperature Modifies Resistance Performances of Rice Varieties to Brown Planthopper,Nilaparvata lugens(Stl)

To investigate the effect of temperature on the resistance characteristics of dce varieties with different resistance genes to brown planthopper （BPH）, Nilaparvata lugens （Stal）, the resistances of IR26 （Bphl） and IR36 （bph2） to BPH population in Hangzhou, China were monitored in greenhouse during September in 2007 and 2008 by using the standard seedling screening techniques （SSST） developed by the International Rice Research Institute （IRRI）. Furthermore, the changes in resistance of IR26 and IR36 to BPH, soluble sugar and oxalic acid contents in 25-day-old rice plants of susceptible variety TN1 and resistant varieties IR26 and IR36 were detected at five temperatures （22℃, 25℃, 28℃, 31℃ and 34℃）. IR26 completely lost resistance both in greenhouse and at the five tested temperatures. IR36 still had moderate resistance at natural temperature, but its resistance decreased gradually when the temperature increased from 25℃ to 34℃, and fully lost its resistance at 31℃ and 34℃. The highest durable resistance of IR26 and IR36 were recorded at 25℃. The soluble sugar content in plants of the three tested rice varieties increased with temperature increase, and the oxalic acid content increased with the temperature increase at first, maximized at 25℃, and then declined. Two-way ANOVA indicated significant effects of temperature and rice variety on contents of soluble sugar and oxalic acid in rice plants

Content Variations of the Secondary Compounds in Rice Plants and Their Influence on Rice Resistance to Brown Planthopper,Nilaparvata lugens

Content variations of the four components in the plants of rice ASD7, IR36 （resistant to brown planthopper） and TN1 （susceptible to brown planthopper）, and their influence on rice resistance to brown planthopper （BPH, Nilaparvata lugens） were investigated. The resistance diversity of rice to BPH biotype II at different plant ages was chiefly attributed to the content variability of the secondary compounds. The contents in the leaf sheath were the lowest as compared with other parts of rice plant. It might explain the reason that BPH has a preference for feeding on leaf sheath aggregately from chemical point of view.