Detection of Rice Yellow Mottle at the Asymptomatic Stage by Hyperspectral Fluorescence and Reflectance Spectroscopies

Rice yellow mottle is considered the most destructive disease threatening rice production in Africa. Early detection of this infection in rice is essential to limit its expansion and proliferation. However, there is no research devoted to the spectral detection of rice yellow mottle virus (RYMV) infection, especially in the asymptomatic or early stages. This work proposes the use of hyperspectral fluorescence and reflectance data at leaf level for the detection of this disease in asymptomatic stages. A greenhouse experiment was therefore conducted to collect hyperspectral fluorescence and reflectance data at different stages of infection. These data allowed to calculate nine vegetation indices: one from fluorescence spectra and eight from reflectance spectra. A t-test made it possible to identify, from the second day after infection, four relevant reflectance vegetation indices to discriminate healthy leaves from those infected: these are Photochemical Reflectance Index (PRI), Transformed Chlorophyll Absorption in Reflectance Index (TCARI), Structure Intensive Pigment Index (SIPI) and Simple Ratio Pigment Index (SRPI). The fluorescence index was less sensitive in detecting infection. The four significant vegetation indices for the detection of RYMV were then used to build and evaluate models for discriminating plants according to their health status by the supervised classification of support vector machine (SVM) at different stages of infection. The maximum overall accuracy is 92.5% six days after inoculation (6 DAI). The sixth day after inoculation would be the adequate day to detect RYMV. This plants discrimination was validated by the mean reflectance spectra and by the histograms showing the differences between the average reflectance vegetation indices values of the two types of plants. Our results demonstrate the feasibility of differentiating RYMV-infected samples. They suggest that support vector machine learning models could be developed to diagnose RYMV-infected plants based on vegetation indices derived from spectral profiles at early stages of disease development.

Study on the Molecular Variation and PCR Detection of Strawberry mottle virus

Strawberry mottle virus （SMoV） is an important viral pathogen infecting strawberry （Fragaria spp.）. The study was conducted to analyze the characterization of the molecular variation of SMoV and develop the methods for detection of SMoV by nested PCR and transcriptional enhancement techniques. The 3 non-coding region （NCR） and large coat protein （LCP） gene of SMoV genome were amplified by reverse transcription-polymerase chain reaction （RT-PCR）. The specific segments were cloned and sequenced. The characterization of the molecular variation for some isolates of SMoV and phylogenetic analysis were studied. Based on the primers located in the conserved region of genome of SMoV, SMoV could be steadily detected using semi-nested PCR and transcriptional enhancement techniques. Both semi-nested PCR and transcriptional enhancement techniques were considerably more sensitive than the standard RT-PCR. The nucleotide sequences of NCR and partial LCP gene of Chinese isolates were obtained, and sequence analysis of the partial LCP gene of various SMoV isolates showed nucleotide identities ranging from 76.8 to 99.7%. There was a slight tendency for isolates to group according to their geographical origin. All 3 Polish isolates, 4 isolates of 7 Dutch isolates, and 3 isolates of 4 Chinese isolates formed a small separate branch, respectively. Two Germanic isolates had a far relationship with other isolates, and formed a separate clade. A high level of sequence variability was found among SMoV isolates, and the Germanic isolates were likely to a special strain group.

Detection and characterization of an isolate of Tomato mottle mosaic virus infecting tomato in China

Tomato（Solanum lycopersicum） plants exhibiting severe leaf distortion, mottle and systemic crinkling symptoms were identified in Hainan province in China in 2016. To survey and control the disease, it is necessary to identify and characterize the pathogen causing the disease. Dot enzyme-linked immunosorbent assay showed that the crude saps of the infected tomato samples reacted positively with the monoclonal antibody against Tobacco mosaic virus which indicated that one or more tobamoviruses are likely associated with the disease. RT-p CR and DNA sequence analysis results further elucidated that Tomato mottle mosaic virus（To MMV） in Tobamovirus was the pathogen causing the mottle disease in tomato. We amplified and sequenced the full-length sequence of the genome which showed the highest nucleotide identity with To MMV YYMLJ and To MMV Ti Lha LJ isolates. The putative virus isolate was named To MMV Hainan. Biological indexing studies showed that To MMV Hainan can infect Nicotiana benthamiana, Capsicum annuum and Solanum lycopersicum showing serious symptoms. This was the first identification and characterization of To MMV infecting tomato in Hainan of China.

First detection and complete genome of Soybean chlorotic mottle virus naturally infecting soybean in China by deep sequencing

Soybean chlorotic mottle virus(SbCMV)was first detected from soybean plants in Jiangxi Province of China by high throughput sequencing and was confirmed by PCR.The complete nucleotide sequence of NC113 was determined to be 8210 nucleotides,and shared the highest similarity(91.7%)with sequences of SbCMV that was only reported in Japan.It encodes nine putative open reading frames(ORFs Ia,Ib and Ⅱ-Ⅷ),and contains a large intergenic region located at nucleotide 5976-6512 between ORFs VI and VII.Sequence analysis and phylogenetic tree indicated that NC113 is an isolate of SbCMV,and is more related to the soymoviruses Blueberry red ringspot virus(BRRSV),Peanut chlorotic streak virus(PCSV)and Cestrum yellow leaf curling virus(CmYLCV)than to other representative members in the Caulimoviridae family.Field survey of 472 legume plants from Jiangxi and Zhejiang provinces showed SbCMV was only detected from soybean in Nanchang City with a low incidence rate.This is the first report of Soybean chlorotic mottle virus identified in China.

Geographical Variation, Distribution and Diversity of <i>Rice Yellow Mottle Virus</i>Phylotypes in Tanzania

Rice yellow mottle virus (RYMV) is the most important disease of rice in Africa. The disease was first observed in1966 inKenya but has now spread in all rice-growing countries of Sub-Saharan Africa. In Tanzania, its distribution has been restricted to the major rice-growing regions. However, the knowledge on RYMV genetic diversity relies on a limited number of coat protein sequences. Previous studies revealed the presence of the phylotypes S4lv, S4lm and strain S5 in Mwanza, Mbeya and Morogoro regions, respectively, and strain S6 in Kilimanjaro region and Pemba Island. Surveys were conducted during the cropping seasons 2013-2014 in eight rice-growing regions of Tanzania to determine geographical variations and phylotypes of RYMV and the influence of environment factors on its distribution and diversity. A total of 185 rice fields were surveyed. Results indicate that prevalence, severity and phylotypes of RYMV varied significantly with rainfall intensity, temperature and relative humidity (P ≤ 0.01). The highest prevalence was found in Morogoro (82%), Mbeya (80%) and Arusha (67.33%) regions whereas Kigoma (9.33%), Rukwa (11.33%) and Shinyanga (18.67%) had the lowest RYMV prevalence. In each region, RYMV prevalence was higher in 2014 than in 2013. The phylotypes S4lm and new determined phylotypes (S6c and S6w) were highly adapted to low temperature (13.3&#176C) and rainfall (13.7 mm) areas. For the first time, strains from the phylotype S4ug were found outside Uganda, in Kilimanjaro region. Strain S4lv (phylotype Lake Victoria) was found for the first time in Arusha region. The strain S4lm was found in Mbeya, Morogoro and Rukwa regions. Strains S4lm and S4lv were also found in Shinyanga and Kigoma regions, respectively. The strain S5 was still restricted to Morogoro but extended to new locations such as Ulanga district. Strain S6 was found in several new areas and new phylotypes of S6 (S6c and S6w) are reported in this study.

Tomato mottle mosaic virus: Characterization, resistance gene effectiveness, and quintuplex RT-PCR detection system

Tomato mottle mosaic virus(ToMMV), an economically important species of the genus Tobamovirus, causes significant loss in yield and quality of tomato fruits. Here, we identified the Shandong isolate of ToMMV(ToMMV-SD) collected from symptomatic tomato fruits in Weifang, Shandong Province of China. ToMMV-SD caused symptoms such as severe mosaic, mottling, and necrosis of tomato leaves, yellow spot and necrotic lesions on tomato fruits. The obtained full genome of ToMMV-SD was 6 399 nucleotides(accession number MW373515) and had the highest identity of 99.5% with that of isolate SC13-051 from the United States of America at the genomic level. The infectious clone of ToMMV-SD was constructed and induced clear mosaic and necrotic symptoms onto Nicotiana benthamiana leaves. Several commercial tomato cultivars, harboring Tm-2~2 resistance gene, and pepper cultivars, containing L resistance gene, were susceptible to ToMMV-SD. Plants of Solanum melongena(eggplant) and Brassica pekinensis(napa cabbage) showed mottling symptoms, while N. tabacum cv. Zhongyan 100 displayed latent infection. ToMMV-SD did not infect plants of N. tabacum cv. Xanthi NN, Brassica rapa ssp. chinensis(bok choy), Raphanus sativus(radish), Vigna unguiculata cv. Yuanzhong 28-2(cowpea), or Tm-2~2 transgenic N. benthamiana. A quintuplex RT-PCR system differentiated ToMMV from tomato mosaic virus, tomato brown rugose fruit virus, tobacco mosaic virus, and tomato spotted wilt virus, with the threshold amount of 0.02 pg. These results highlight the threat posed by ToMMV to tomato and pepper cultivation and offer an efficient detection system for the simultaneous detection of four tobamoviruses and tomato spotted wilt virus infecting tomato plants in the field.

Construction of chimeric viruses based on pepper mild mottle virus using a modiffed Cre/loxP system

Cre/loxP,a site-specific recombination system,has been widely used for various purposes,including chromosomal translocations,generation of marker-free transgenic plants,tissue-specific activation of a reporter gene and efficient heterologous gene expression in plants.However,stable or transient expression of Cre recombinase in plants can cause chlorosis or necrosis.Here,we describe a modified Cre/loxP recombination system using a DNA fragment flanked with loxP sites in the same orientation in which necrosis induced by Cre recombinase in Nicotiana benthamiana leaves was alleviated.The modified system was successfully used to create functional GFP-tagged pepper mild mottle virus(PMMoV)and a chimeric virus with coat protein(CP)substitution assembled from separate pro-vector modules.Our results provide a new strategy and flexible technique to construct chimeric virus and infectious clones for plant viruses with large genomes.

Mapping subgenomic promoter of coat protein gene of Cucumber green mottle mosaic virus

Many plant viruses utilize subgenomic RNA as gene expression strategy, therefore mapping subgenomic promoter(SGP) is extremely important for constructing viral vectors. Although Cucumber green mottle mosaic virus(CGMMV)-based virus vectors have been constructed, SGP of the coat protein(CP) has not yet mapped. To this end, we firstly presumed 13 nucleotides upstream of the start codon as the transcription starting site(TSS) as previous study identified by random amplification of c DNA ends(RACE). Secondly, the region from nucleotides –110 to +175 is the putative CP SGP, as predicted, a long stem loop structure by the secondary structure of RNA covering movement protein(MP) and CP. To map the CGMMV CP SGP, we further constructed a series of deletion mutants according to RNA secondary structure prediction. The deletion of TSS upstream significantly enhanced CP transcription when 105 nucleotides were retained before the CP TSS. For the downstream of CP TSS, we analyzed the expression of enhanced green fluorescent protein(EGFP) in a series of vectors with partial deletion of the CGMMV CP and found that the nucleotides from +71 to +91 played a key role in the EGFP expression at the transcription level, while EGFP showed the highest expression level when 160 nucleotides were retained downstream of the CP TSS. To confirm these results, we applied online software MEME to predict the motifs and cis-acting elements in the 466 nucleotides covering the sequences of deletion analysis. Conserved motifs and relative acting elements were in regions in which transcription levels were the highest or enhanced. To our best knowledge, this is the first mapping of CGMMV SGP.

Impact of <i>Cowpea mottle virus</i>on the Growth and Yield of Bambara Groundnut (<i>Vigna subterranean</i>(L.) Verdc.)

Bambara groundnut [Vigna subterranean (L.) Verdc] is a food legume with high potential for food security in Sub-Saharan Africa. However, in addition to being a neglected crop, its production is limited by several constraints among which viral diseases are most cited. In order to contribute to the improvement of Bambara groundnut in Burkina Faso, local accessions of the crop were screened for resistance to Cowpea mottle virus (CPMoV), one of the most damaging viruses in grain legumes. Seven local accessions (C1 to C7) from two agro-ecological zones were evaluated by mechanical inoculation in field conditions in 2016 and 2017. The infected plants exhibited various symptoms of chlorosis, leaf deformation, growth retardation and plant stunting. CPMoV caused a significant reduction in the number of flowers and pods. As a result, grain yield was reduced by 49.5% to 83.9% depending on the accessions. The impact of the virus in yield loss was lowest in accessions C6 and C7 which indicated their possible used in the management of Cowpea mottle virus disease in bambara groundnut.

An Attenuated Strain of Cucumber Green Mottle Mosaic Virus as a Biological Control Agent against Pathogenic Viral Strains

Cucumber green mottle mosaic virus (CGMMV), a member of the Tobamovirus genus, causes a severe disease of cucurbits. In the Moscow region of Russian Federation, the incidence of infection on cucumber plants in greenhouses is high;however, the virus is poorly studied. In this work, the full-length genomes of two pathogenic MC-1 and MC-2 strains of CGMMV isolated from cucumber plants grown in greenhouses in the Moscow region and the attenuated VIROG-43M strain were sequenced. Comparison of VIROG-43M nucleotide sequence with those of the pathogenic strains revealed three missense mutations. Their role in attenuation is discussed. For the first time, in a number of trials conducted under laboratory conditions and in commercial greenhouses, the efficiency of the attenuated VIROG-43M strain as a biocontrol agent for cucumber plant protection resulting in significant yield gain was demonstrated. Phylogenetic analysis with 83 full-length CGMMV coat protein genes isolated in 16 different countries showed that Russian strains are related to isolates from Spain, Greece, USA and Israel.

Monoclonal antibody-based serological methods for maize chlorotic mottle virus detection in China

Maize chlorotic mottle virus (MCMV) infects maize plants and causes significant losses in corn production worldwide. In this study, purified MCMV particles were used as the immunogen to produce monoclonal antibodies (MAbs) and polyclonal antibodies (PAbs). Four murine MAbs (4B8, 8C11, 6F4, and 9G1) against MCMV were obtained through the hybridoma technology. The triple antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA), dot-immunobinding assay (DIBA), and immunocapture reverse transcription-polymerase chain reaction (IC-RT-PCR) using the MAb 4B8 were then developed for sensitive, specific, and rapid detection of MCMV in fields. MCMV could be detected in infected leaf crude extracts at dilutions of 1:327 680, 1:64000, and 1:3276800 (w/v, g/ml) by TAS-ELISA, DIBA, and IC-RT-PCR, respectively. One hundred and sixty-one maize field samples showing virus-like symptoms and sixty-nine symptomless maize field samples from ten different provinces of China were collected and screened for the presence of MCMV using the established serological methods. A phylogenetic tree was constructed based on the full length CP genes and Chinese MCMV isolates formed one branch with Thailand isolates. The detection results demonstrated that MCMV is one of most prevalent viruses infecting maize in the Yunnan and Sichuan provinces of China.

High-throughput Sequencing Reveals vsiRNAs Derived from Cucumber green mottle mosaic virus-infected Watermelon

Cucumber green mottle mosaic virus(CGMMV) is a member of the genus Tobamovirus, and is a serious pathogen of Cucurbitaceae crops. Virusderived small interfering RNAs(vsi RNAs), which are processed by Dicer-like and Argonaute proteins as well as RNA-dependent RNA polymerase,mediate the silencing of viral genomic RNA and host transcripts. To identify the CGMMV derived vsi RNAs and reveal interactions between CGMMV and watermelon host plant, deep sequencing technology was used to identify and characterize the vsi RNAs derived from CGMMV in infected watermelon plants in present study. A total of 10 801 368 vsi RNA reads representing 71 583 unique s RNAs were predicted in CGMMVinoculated watermelon plants. The CGMMV vsi RNAs were mostly 21 or 22 nt long. The majority of the CGMMV vsi RNAs(i.e., 91.7%) originated from the viral sense strand. Additionally, uracil was the predominant 5′-terminal base of vsi RNAs. Furthermore, the putative targets and functions of some of the CGMMV vsi RNAs were predicted and investigated. The results enhance our understanding of the interaction between CGMMV and the host watermelon and provide molecular basis for CGMMV resistance improvement in watermelon and other Cucurbitaceae crops.

Decay of amplitude-modulation property of song and an analysis of sounding mechanism in mottled cicade (Gaeana maculata Drury)

In the natural song of the mottled cicada, the prelude and loud song of the prolonging and repctition songs consist separatcly of several pulses containing the three subpulscs (SP1-3). The principal frequencies (PFband PFc) of the basic sound and carricr frcquency band are 338±20 Hz and 3617±87 Hz, respectively.The prelude or the prolonging song contains 22 pulses (P1-22),and their amplitude-modulation propertics gradually decay. The carrier-wave oscillations gradually lose symmetrics and overlap with basic waves of the SP1, and the SP2,3 are similar to sine waves. Moreover, a dominant tone of the song gradually changes from PFc into PFb. In the loud song of the prolonging song, the prelude and loud song of repetition songs, the amplitude-modulation propcrties are lost.The carrier-wave oscillations mainly overlap with basic waves of the SP2, and the amplitudes of the PFc arc 10 dB over lower than those of the PFb, and the songs are the PFb of basic sounds as a dominant tone. In view of the structure of the sounding membrane, the vibration frequencies of the three long ribs may be gradually changed from mass controlling to mechanical resistance controlling, and corresponding songs are gradually changed from the prolonging prelude with the PFc as a dominant tone to the loud song and repetition songs with the PFb as a dominant tone.

侵染丝瓜的黄瓜绿斑驳花叶病毒山东分离物分子鉴定

2013年,在山东泰安采集到疑似感染黄瓜绿斑驳花叶病毒(Cucumber green mottle mosaic virus,CGMMV)的丝瓜样品,利用该病毒外壳蛋白(Coat protein,CP)特异引物CG-CPF/CPR对样品进行扩增并测序。所得序列(GenBank登录号:KJ187042)经NCBI BLAST比对,与已登录的CGMMV序列的相似性均大于92.6%,确定为黄瓜绿斑驳花叶病毒。系统进化分析表明,CGMMV山东丝瓜分离物(CGMMV-SDLoofah)与辽宁分离物(CGMMV-LN)等大多数国内CGMMV分离物同聚为一个进化组,从而进一步确定了CGMMV山东丝瓜病毒分离物为黄瓜绿斑驳花叶病毒。这是首次报道黄瓜绿斑驳花叶病毒侵染丝瓜。

Recent progress in maize lethal necrosis disease:From pathogens to integrated pest management

Maize(Zea mays),as a staple food and an important industrial raw material,has been widely cultivated for centuries especially by smallholder farmers.Maize lethal necrosis disease(MLND)is a serious disease infecting maize,which caused devastating damage in the African region recently.MLND is induced by co-infection of maize chlorotic mottle virus and one of several cereal-infecting viruses in the Potyviridae family,with the symptoms ranging from chlorotic mottle to plant death at different infection stages.Integrated pest management for MLND needs strengthening detection,focusing on prevention and effective control.Early detection system of MLND has been successfully established by serological methods,nucleic acid-based methods,next-generation sequencing,etc.The practices,such as using certified seeds,sanitary measures,crop rotation,tolerant or resistant varieties etc.,have been considered as the effective,economical and eco-friendly way to prevent and control MLND.

盐城地区开展黄瓜绿斑驳花叶病毒病识别与防控的主要措施

黄瓜绿斑驳花叶病毒(cucumber green mottle mosaic virus,CGMMV)属芜菁花叶病毒科(Tymoviridae)烟草花叶病毒属(Tobamovirus),是葫芦科作物瓜类上发生的一种病毒病。CGMMV主要通过种子带毒传播,也可通过接触传染,为国际上一种重要的检疫性植物病毒。已经报道的CGMMV寄主有黄瓜、西瓜、甜瓜等葫芦科植物。1935年Ainsworth在英国首次发现该病毒,此后在欧洲传播,60年代因引种传入到亚洲的日本等国,80年代传入我国台湾。

Molecular Variability and Genetic Structure of IYMV in Burkina Faso

Imperata yellow mottle virus (IYMV, Sobemovirus) was first described in 2008 in the south-western region of Burkina Faso (West Africa). The genetic diversity of IYMV was not documented up to day. In this study, the variability of CP of IYMV was evaluated through the molecular characterization of 38 isolates collected in the western part of Burkina Faso. Comparison of sequences of these new isolates and one IYMV sequence available in GenBank revealed that the average nucleotide diversity was low. The ratio of non-synonymous over synonymous nucleotide substitutions per site was low, indicating a CP diversification under strong purifying selection. Despite of the low nucleotide diversity, phylogenetic analyses revealed segregation of IYMV isolates into six major clades. There was no correlation of phylogenetic grouping of isolates based on geographical location. This is the first study of the genetic diversity of IYMV.

Tobacco Vein Mottling Virus完整基因组上串联重复序列分布

利用自编计算机程序提取并展示Tobacco Vein Mottling Virus完整基因组上串联重复序列分布特性,从而为有效利用串联重复序列分布特性研究完整基因组的结构和功能、遗传和变异规律提供完备、细仔的数据支撑。

Genetic Analysis and Molecular Mapping of Two SMV-Resistance Traits in Soybean:Adult-Plant Resistance and Resistance to Seed Coat Mottling

Soybean mosaic virus （SMV） could lead to adult-plant system diseases, and cause mottling of soybean seeds. Genetic analysis and molecular mapping were conducted using an F2 population and derived F3 families from two crosses of Dongnong 3C624 （susceptible）x Dongnong 8143 （resistant） and Dongnong 3C628 （susceptible）× Tie 6915 （resistant）. Simple sequence repeat （SSR） markers with bulked segregation analysis （BSA） were used to conduct genetic mapping of the resistance to SMV1 in the segregating populations. The results indicated that resistance to SMV1 in adultplants and the resistance to seed coat mottling in Dongnong 8143 and Tie 6915 was separately controlled by one single dominant gene. The two dominant genes were identified to be linked on the MLG F by Mendel＇s genetics and SSR genetic mapping. The order and distance of markers DPRSMV1 and DSRSMV1 were Sat 229-6.9 cM-DSRSMV1-4.6 cM-Sat_317-3.6 cM-DPRSMV1-5.2 cM-Satt335. The order and distance of markers TPRSMV1 and TSRSMV1 was Satt160-16.1 cM-TPRSMV1-7.3 cM-Satt516-2.0 cM-TSRSMV1-4.5 cM-Sat_133. This research provides the useful information for breeders to select the two types of SMV resistance simultaneously in soybean breeding through molecular marker-assisted selection （MAS）.