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Maize rough dwarf disease (MRDD) is a viral disease that causes substantial yield loss, especially in China's summer planted maize area. Discovery of resistance genes would help in developing high-yielding resistant maize hybrids. Genome-wide association studies (GWASs) have advanced quickly and are now a powerful tool for dissecting complex genetic architectures. In this study, the disease severity index (DSI) of 292 maize inbred lines and an F 6 linkage population were investigated across multiple environments for two years. Using the genotypes obtained from the Maize SNP 50K chip, a GWAS was performed with four analytical models. The results showed that 22 SNPs distributed on chromosomes 1, 3, 4, 6, 7 and 8 were significantly associated with resistance to MRDD (P<0.0001). The SNPs on chromosomes 3, 6 and 8 were consistent with the quantitative trait locus (QTL) regions from linkage mapping in an RIL population. Candidate genes identified by GWAS included an LRR receptor-like serine/threonine-protein kinase (GRMZM2G141288), and a DRE-binding protein (GRMZM2G006745). In addition, we performed an allele variation analysis of the SNP loci selected by GWAS and linkage mapping and found that the main alleles of the two SNP loci PZE&#95;101170408 and PZE&#95;106082685 on chromosome 1 differed in terms of disease-resistant materials and disease-susceptible materials. The identified SNPs and genes provide useful information for MRDD-related gene cloning and insights on the underlying disease resistance mechanisms, and they can be used in marker-assisted breeding to develop MRDD-resistant maize., (Copyright © 2021. Published by Elsevier B.V.)

Typically associated with fungal species, members of the viral family Totiviridae have recently been shown to be associated with plants, including important crop species, such as Carica papaya (papaya) and Zea mays (maize). Maize-associated totivirus (MATV) was first described in China and more recently in Ecuador, where it has been found to co-occur with other viruses known to elicit maize lethal necrosis disease (MLND). In a survey for maize-associated viruses, 35 samples were selected for Illumina HiSeq sequencing, from the Tanzanian maize producing regions of Mara, Arusha, Manyara, Kilimanjaro, Morogoro and Pwani. Libraries were prepared using an RNA-tag-seq methodology. Taxonomic classification of the resulting datasets showed that 6 of the 35 samples from the regions of Arusha, Kilimanjaro, Morogoro and Mara, contained reads that were assigned to MATV reference sequences. This was confirmed with PCR and Sanger sequencing. Read assembly of the six MATV-associated datasets yielded partial MATV genomes, two of which were selected for further characterization, using RACE. This yielded two full-length MATV genomes, one of which is divergent from other available MATV genomes.

Background: Rice black-streaked dwarf virus (RBSDV) and Southern rice black-streaked dwarf virus (SRBSDV) seriously interfered in the production of rice and maize in China. These two viruses are members of the genus Fijivirus in the family Reoviridae and can cause similar dwarf symptoms in rice. Although some studies have reported the phylogenetic analysis on RBSDV or SRBSDV, the evolutionary relationship between these viruses is scarce., Methods: In this study, we analyzed the evolutionary relationships between RBSDV and SRBSDV based on the data from the analysis of codon usage, RNA recombination and phylogenetic relationship, selection pressure and genetic characteristics of the bicistronic RNAs (S5, S7 and S9)., Results: RBSDV and SRBSDV showed similar patterns of codon preference: open reading frames (ORFs) in S7 and S5 had with higher and lower codon usage bias, respectively. Some isolates from RBSDV and SRBSDV formed a clade in the phylogenetic tree of S7 and S9. In addition, some recombination events in S9 occurred between RBSDV and SRBSDV., Conclusions: Our results suggest close evolutionary relationships between RBSDV and SRBSDV. Selection pressure, gene flow, and neutrality tests also supported the evolutionary relationships.

Maize chlorotic mottle virus (MCMV) was first reported in maize in China in 2009. In this study we further analyzed the epidemiology of MCMV and corn lethal necrosis disease (CLND) in China. We determined that CLND observed in China was caused by co-infection of MCMV and sugarcane mosaic virus (SCMV). Phylogenetic analysis using four full-length MCMV cDNA sequences obtained in this study and the available MCMV sequences retrieved from GenBank indicated that Chinese MCMV isolates were derived from the same source. To screen for maize germplasm resistance against MCMV infection, we constructed an infectious clone of MCMV isolate YN2 (pMCMV) and developed an Agrobacterium-mediated injection procedure to allow high throughput inoculations of maize with the MCMV infectious clone. Electron microscopy showed that chloroplast photosynthesis in leaves was significantly impeded by the co-infection of MCMV and SCMV. Mitochondria in the MCMV and SCMV co-infected cells were more severely damaged than in MCMV-infected cells. The results of this study provide further insight into the epidemiology of MCMV in China and shed new light on physiological and cytopathological changes related to CLND in maize.

Background: Sugarcane mosaic virus (SCMV) is responsible for large-scale economic losses in the global production of sugarcane, maize, sorghum, and some other graminaceous species. To understand the evolutionary mechanism of SCMV populations, this virus was studied in Shanxi, China. A total of 86 maize leaf samples (41 samples in 2012 and 45 samples in 2013) were collected from 4 regions of Shanxi., Results: Double-antibody sandwich (DAS)-ELISA and RT-PCR showed 59 samples (30 samples in 2012 and 29 samples in 2013) to be positive for SCMV, from which 10 new isolates of SCMV were isolated and sequenced. The complete genomes of these isolates are 9610 nt long, including the 5' and 3' non-coding regions, and encode a 3063-amino acid polyprotein. Phylogenetic analyses revealed that 24 SCMV isolates could be divided on the basis of the whole genome into 2 divergent evolutionary groups, which were associated with the host species. Among the populations, 15 potential recombination events were identified. The selection pressure on the genes of these SCMV isolates was also calculated. The results confirmed that all the genes were under negative selection., Conclusions: Negative selection and recombination appear to be important evolutionary factors shaping the genetic structure of these SCMV isolates. SCMV is distributed widely in China and exists as numerous strains with distinct genetic diversity. Our findings will provide a foundation for evaluating the epidemiological characteristics of SCMV in China and will be useful in designing long-term, sustainable management strategies for SCMV.

Deep sequencing of small RNA (sRNA) populations in maize plants from southwest China resulted in the identification of a previously unknown dsRNA virus with a sequence and genome organization resembling that of a totivirus. The complete viral genome is 3,956 nucleotides in length and contains two open reading frames (ORFs) with the potential to produce a ORF1-ORF2 fusion protein through a -1 ribosomal frameshift translation mechanism. ORF1 encodes the putative capsid protein (CP), whereas the predicted product of ORF2 contains motifs typical of an RNA-dependent RNA polymerase (RdRp). Phylogenetic analysis using the amino acid sequences of putative RdRp fusion proteins showed that the new virus was grouped in a clade together with the totiviruses, suggesting that it is a new member of the genus Totivirus of the family Totiviridae. The virus is tentatively named "maize-associated totivirus (MATV)". Our findings demonstrate that it is feasible to identify totiviruses by deep sequencing of small RNAs.

Background: Maize streak Reunion virus (MSRV) is a member of the Mastrevirus genus in the family Geminiviridae. Of the diverse and increasing number of mastrevirus species found so far, only Wheat dwarf virus and Sweetpotato symptomless virus 1 have been discovered in China. Recently, a novel, unbiased approach based on deep sequencing of small interfering RNAs followed by de novo assembly of siRNA, has greatly offered opportunities for plant virus identification., Methods: Samples collected from maize leaves was deep sequencing for virus identification. Subsequently, the assay of PCR, rolling circle amplification and Southern blot were used to confirm the presence of a mastrevirus., Results: Maize streak Reunion virus Yunnan isolate (MSRV-[China:Yunnan 06:2014], abbreviated to MSRV-YN) was identified from maize collected from Yunnan Province, China, by small RNA deep sequencing. The complete genome of this virus was ascertained as 2,880 nucleotides long by conventional sequencing. A phylogenetic analysis showed it shared 96.3 % nucleotide sequence identity with the isolate of Maize streak Reunion virus from La Reunion Island. To our knowledge, this is the first identification of MSRV in China. Analyses of the viral derived small interfering RNAs (vsiRNAs) profile showed that the most abundant MSRV-YN vsiRNAs were 21, 22 and 24 nt long and biased for A and G at their 5' terminal residue. There was a slightly higher representation of MSRV-YN siRNAs derived from the virion-sense strand genome than the complementary-sense strand genome. Moreover, MSRV-YN vsiRNAs were not uniformly distributed along the genome, and hotspots were detected in the movement protein and coat protein-coding region., Conclusions: A mastrevirus MSRV-YN collected in Yunnan Province, China, was identified by small RNA deep sequencing. This vsiRNAs profile derived from MSRV-YN was characterized, which might contribute to get an insight into the host RNA silencing defense induced by MSRV-YN, and provide guidelines on designing antiviral strategies using RNAi against MSRV-YN.

Rice black-streaked dwarf virus (RBSDV) is an economically important virus that causes maize rough dwarf disease and rice black-streaked dwarf disease in East Asia. To study RBSDV variation and recombination, we examined the segment 9 (S9) sequences of 49 RBSDV isolates from maize and rice in China. Three S9 recombinants were detected in Baoding, Jinan, and Jining, China. Phylogenetic analysis showed that Chinese RBSDV isolates could be classified into two groups based on their S9 sequences, regardless of host or geographical origin. Further analysis suggested that S9 has undergone negative and purifying selection.

Rice black-streaked dwarf virus (RBSDV) is a double-stranded RNA (dsRNA) virus belonging to the genus Fijivirus in the family Reoviridae. The genome of RBSDV consists of ten dsRNA segments. Although RBSDV has caused significant economic losses to rice and maize production in the past few years in China, its molecular diversity and evolution remain largely unknown. To elucidate the factor(s) underlying the evolution of RBSDV, we determined segment 8 (S8; carrying ORF8 encoding the minor core capsid protein) sequences of 101 samples and segment 10 (S10; carrying ORF10 encoding the major capsid protein) sequences of 103 samples. The results show that both ORF8 and ORF10 are under negative selection. The S8 of three isolates and S10 of two isolates are recombinants. The RBSDV population in China can be classified into three groups according to S8 sequences or into two groups according to S10 sequences, irrespective of host or geographical origin. Of the RBSDV isolates with both S8 and S10 sequences available, 17 are between-group reassortants and 30 are between-subgroup reassortants. The RBSDV subpopulations from different geographical regions and hosts show frequent gene flow within or between subpopulations. The RBSDV population from maize is in a state of expansion. In this study, no new emergent population was detected. Taken together, the results indicate that, in addition to recombination and negative selection, reassortment and gene flow are important factors that drive evolution of RBSDV in China.

Background: Rice and maize dwarf diseases caused by the newly introduced Southern rice black-streaked dwarf virus (SRBSDV) have led to severe economic losses in South China in recent years. The distribution and diversity of SRBSDV have not been investigated in the main rice and maize growing areas in China. In this study, the distribution of SRBSDV in China was determined by using reverse transcription-polymerase chain reaction (RT-PCR)., Results: Between 2009 and 2010, 2404 plant samples (2294 rice, 110 maize samples, and more than 300 cultivars) with dwarf symptoms were collected from fields in 194 counties of 17 provinces in China and SRBSDV was detected. The results indicated that 1545 (64.27%) of samples (both rice and maize) were infected with SRBSDV. SRBSDV was detected widely in Hainan, Guangdong, Guangxi, Yunnan, Guizhou, Chongqing, Fujian, Jiangxi, Hunan, Hubei, Anhui, Jiangsu, and Zhejiang provinces, which suggests SRBSDV is an important pathogen causing rice dwarfing diseases in South China. Phylogenetic analysis of 15 representative virus isolates revealed that SRBSDV isolates in China had high levels of nucleotide and amino acid sequence identities (>97.8%)., Conclusions: SRBSDV spreads naturally in Yangtze River basin and south region, the location of the major rice production areas. In comparison, the virus rarely spreads north of Yangtze River in North China. Distribution of SRBSDV is consistent with the migrating and existing ranges of its vector WBPH, suggesting that SRBSDV might be introduced into South China along with the migration of viruliferous WBPH.

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 1:327680, 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.

Southern rice black-streaked dwarf virus (SRBSDV) is a novel Fijivirus prevalent in rice in southern and central China, and northern Vietnam. Its genome has 10 segments of double-stranded RNA named S1 to S10 according to their size. An isolate of SRBSDV, JNi4, was obtained from naturally infected maize plants from Ji'ning, Shandong province, in the 2008 maize season. Segments S7 to S10 of JNi4 share nucleotide identities of 72.6%-73.1%, 72.3%-73%, 73.9%-74.5% and 77.3%-79%, respectively, with corresponding segments of Rice black-streaked dwarf virus isolates, and identities of 99.7%, 99.1%-99.7%, 98.9%-99.5%, and 98.6%-99.2% with those of SRBSDV isolates HN and GD. JNi4 forms a separate branch with GD and HN in the phylogenetic trees constructed with genomic sequences of S7 to S10. These results confirm the proposed taxonomic status of SRBSDV as a distinct species of the genus Fijivirus and indicate that JNi4 is an isolate of SRBSDV. Shandong is so far the northernmost region where SRBSDV is found in China.

The complete nucleotide sequence of the prevalent strain of a potyvirus isolated from maize in Beijing, China was determined and compared with other closely related potyviruses. The viral genome comprises 9595 nucleotides, excluding the poly (A) tail, and encodes a putative polyprotein of 3063 amino acid residues. Sequence comparison of the coat proteins showed that this isolate was most closely related to most other potyviral isolates infecting maize across China with identities of about 99% and thus represented the prevalent strain. It was also closely related to most isolates of Sugarcane mosaic virus (SCMV) infecting maize in Europe with maximum identity of about 95% at the amino acid level. The polyprotein sequence of the Beijing isolate shares identities of 98% with those of two other Chinese maize isolates and shares identity of 69% with Maize dwarf mosaic virus-Bulgarian isolate, respectively. Phylogenetic analyses of the sequences indicated that the Beijing isolate can be tentatively referred to as a prevalent strain of SCMV.

The full-length nucleotide sequence of a potyvirus causing the maize dwarf mosaic (MDM) disease in Henan province, central China, was obtained by reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of the cDNA 5'-end (5'-RACE). The viral genome comprised of 9596 nucleotides except the polyA tail and encoded a putative polyprotein of 3603 amino acids. The entire genomic sequence of this isolate shared identities of 94.2% and 98.3% with Sugarcane mosaic virus (SCMV) HZ isolate at the nucleotide and deduced amino acid levels, respectively, but only a 69.1% identity with MDM virus (MDMV) Bulgarian isolate (MDMV-Bg) at the nucleotide level. Phylogenetical tree analysis of the complete nucleotide sequences indicated that the Henan isolate of a potyvirus causing MDM disease is in fact a Henan strain of SCMV (SCMV-HN).

Sugarcane mosaic virus (SCMV) was detected in all 62 maize samples collected from eight maize-growing provinces in China showing dwarf mosaic symptoms by immunocapture reverse-transcription polymerase chain reaction (RT-PCR). Maize dwarf mosaic virus (MDMV), Sorghum mosaic virus (SrMV) and Johnsongrass mosaic virus (JGMV), however, were not detected in any of the samples by RT-PCR. Eleven cDNA fragments of approximately 0.8 kilobases covering most of the coat protein (CP) gene of SCMV were sequenced and sequence analysis indicates that these eleven isolates share 98.1 to 100 % identity at the amino acid level. Sequence comparison and phylogenetic analysis of the CP genes from the eleven Chinese isolates as well as 21 SCMV subgroup virus isolates indicate that the eleven Chinese virus isolates were closely related to SCMV with 97.0 to 98.1 % sequence identity at the amino acid level, while relatively lower sequence identity was found with MDWV, SrMV or JGMV. The results indicate that the Chinese isolates are members of the SCMV species, and thus, SCMV can be considered as the most common and important potyvirus infecting maize in China.

A viral disease with dwarfing symptoms is associated with severe damage of different cereal crops including rice, maize, wheat and sorghum grown in China. It is believed that the pathogenic agent of the disease on rice and sorghum is rice black streaked dwarf virus (RBSDV), however, the cause of maize dwarf disease in China is still inconclusive. In this report, dsRNA was isolated from virus particles obtained from the diseased plants of rice, maize, wheat and sorghum from two Chinese provinces. Full-length cDNAs of genome segments 9 (S9) and 10 (S 10) were obtained through a RT-PCR approach. Sequence analysis showed that the S9 sequences of Chinese isolates and Japanese RBSDV isolate were very similar to each other (89.1-89.6% identity at the nucleotide level, 92.3-92.9% and 95.8-98.6% identity at the amino acid level for ORF1 and ORF2, respectively). In addition, the S10 sequences of Chinese isolates and Japanese RBSDV were very similar to each other (93.0-95.4% identical nucleotides and 96.2-97.0% identical amino acids, respectively). However, there were lower similarities for S9 and S10 sequences between Chinese isolates and an Italian Maize Rough Dwarf Virus (MRDV) isolate. Phylogenetic analysis indicates that Chinese viral isolates found to infect rice, maize, wheat and sorghum and leading to similar cereal dwarfing manifestations could be grouped to the same virus species, RBSDV.

Sugarcane or maize leaves with mosaic virus symptoms were collected from 13 sites in China. Sequence data showed that all 8 samples from maize contained Sugarcane mosaic virus (SCMV); complete sequences were determined from 2 samples and partial sequences (the CI coding region and the 3'-part of the genome) from the others. The 5 sugarcane samples all contained a virus tentatively described as Sorghum mosaic virus (SrMV) and in three of them SCMV was also detected; 2 SrMV sequences and the 3 SCMV ones were completely determined and partial SrMV sequences were obtained from the remaining 3 samples. The features of the complete sequences of SCMV and SrMV are described for the first time. Sequence comparisons and phylogenetic analysis showed three distinct groups of Chinese SCMV sequences (sugarcane isolates from Zhejiang province, a maize isolate from Guangdong and maize isolates from other provinces). The SrMV sequences were similar to one another (> 93% identical nucleotides); they resembled published sequences in the coat protein but were less similar in the 3'-UTR. The complete sequences of SCMV and SrMV had about 70% nucleotides identical to one another and to Maize dwarf mosaic virus (MDMV). MDMV was not detected in any of the samples.

A universal primer (Sprimer: 5'-GGX AAY AAY AGY GGX CAZ CC-3', X = A, G, C or T; Y = T or C; Z = A or G), designed from the consensus sequences that code for the conserved sequence GNNSGQP in the NIb region of members of the family Potyviridae, was used to amplify by RT-PCR the 3'-terminal genome regions from infected plant samples representing 21 different viruses in the family. Sequencing of some of the fragments (c. 1.7 kb) showed that the type strain (ATTC PV-107) of Oat necrotic mottle virus is not a distinct species in the genus Rymovirus, but is synonymous with Brome streak mosaic virus (genus Tritimovirus) and that Celery mosaic virus is a distinct member of the genus Potyvirus not closely related to any other sequenced species. Potyviruses infecting crops in China were also investigated, showing that viruses on cowpea and maize in Hangzhou, Zhejiang province were respectively Bean common mosaic virus and Sugarcane mosaic virus and that one on garlic in Nanjing, Jiangsu province was Onion yellow dwarf virus. Fragments were also sequenced from Chinese isolates of Lettuce mosaic virus and Soybean mosaic virus (from Hangzhou), Turnip mosaic virus (2 different isolates from Zhejiang province) and RNA1 of Wheat yellow mosaic virus (from Rongcheng, Shandong province).

Three virus isolates from maize with rough dwarf in different provinces in China were analyzed at the molecular level. When compared to an isolate from diseased rice plants in Hubei Province, all four isolates had identical genomic RNA electrophoretic profiles, which were composed of ten double-stranded (ds) RNAs. Full-length cDNAs of segment 10 (S10) from each of the four isolates were cloned by RT-PCR and the complete sequences were determined. Analysis of the sequences revealed that each consisted of 1801 nucleotides and contained a single open reading frame (ORF) which potentially encoded a protein with 558 amino acids. Further, the sequences showed more than 97.0% and 98.0% identity at nucleotide and amino acid levels, respectively. In addition, their identities to rice black-streaked dwarf virus S10 were significantly higher than those to maize rough dwarf virus S10. Based on these results, it is suggested that the virus which causes this maize disease in China is rice black-streaked dwarf virus.