Bacilliform Virus Research Articles

Complete genome of the new bacilliform virus that causes Milky Hemolymph Syndrome in Chionoecetes bairdi (Rathbun, 1924)

The genome of a new member of the Nimaviridae family has been sequenced. The Chionoecetes bairdi bacilliform virus (CbBV) causes Milky Hemolymph Syndrome (MHS) in Chionoecetes bairdi populations of the Pacific coast of Kamchatka. The CbBV genome is represented by double-stranded DNA with a length of 245,567 nucleotides containing 120 ORFs. Of these, 85 proteins had significant matches in the NCBI database, and 57 genes encoded capsid, envelope, tegument and nonstructural proteins. Comparative analysis of the genomes of CbBV and a number of representatives of the class nuclear arthropod large DNA viruses (NALDVs) made it possible to isolate 49 evolutionarily conserved orthologue core genes. Among them, 5 were multicopy genes, and 44 were single-copy genes. There were ancestral genes characteristic of all Naldaviricetes − per os infectivity complex genes, one DNA polymerase gene and one thymidylate synthase gene.Phylogenetic analysis of representatives of the Nimaviridae family revealed that the CbBV and Chionoecetes opilio bacilliform virus (CoBV) form an independent clade within the family separate from the clade containing WSSV strains. This is supported by data on the order and arrangement of genes in the genomes of nimaviruses that were identical within each clade but differed between them. In addition, a high identity of the genomes and proteomes of CbBV and CoBV (approximately 99%) was shown, and their identity with WSSV strains was no more than 33%.The data on the structure of the genome of the new virus that causes MHS in C. bairdi indicate that it belongs to the family Nimaviridae, genus Whispovirus. Thus, the CbBV infecting the commercially important species of Tanner crab in populations of the Pacific coast of Kamchatka is the second “wild” representative of replicating nimaviruses whose genome has been characterized after the CoBV that causes MHS in C. opilio in populations of the Sea of Japan. The discovery of a new member of the family that infects decapods indicates the prevalence of nimaviruses in marine ecosystems. The information obtained is important for understanding the evolution of representatives of the class of nuclear arthropod large DNA viruses. The discovery of a new nimavirus that causes MHS in Chionoecetes crabs, in contrast to the white spot syndrome (WSS) caused by WSSV strains, makes it relevant to identify two variants and possibly species within the family, namely, WSSV and Milky Hemolymph Syndrome virus (MHSV).

Analysis of plant pararetrovirus promoter sequence(s) for developing a useful synthetic promoter with enhanced activity in rice, pearl millet, and tobacco plants.

Promoters are one of the most important components for many gene-based research as they can fine-tune precise gene expression. Many unique plant promoters have been characterized, but strong promoters with dual expression in both monocot and dicot systems are still lacking. In this study, we attempted to make such a promoter by combining specific domains from monocot-infecting pararetroviral-based promoters sugarcane bacilliform virus (SCBV) and banana streak virus (BSV) to a strong dicot-infecting pararetroviral-based promoter mirabilis mosaic virus (MMV). The generated chimeric promoters, MS, SM, MB, and BM, were tested in monocot and dicot systems and further validated in transgenic tobacco plants. We found that the developed chimeric promoters were species-specific (monocot or dicot), which depended on their respective core promoter (CP) region. Furthermore, with this knowledge, deletion-hybrid promoters were developed and evaluated, which led to the development of a unique dual-expressing promoter, MSD3, with high gene expression efficiency (GUS and GFP reporter genes) in rice, pearl millet, and tobacco plants. We conclude that the MSD3 promoter can be an important genetic tool and will be valuable in plant biology research and application.

Detection of Rice Tungro Bacilliform Virus (RTBV) causes of tungro disease in rice plant (Oryza sativa l.) in Langkat, North Sumatera using PCR (Polymerase Chain Reaction) technique

Tungro is one of the important disease in rice which is an obstacle in increasing rice production in Langkat. The detection of diseases caused by this virus has many barrier since there are many virus also have similar symptoms in rice plants, therefore it requires a more accurate technique like PCR. The PCR detects directly the virus itself or its type. Rice leaf samples were taken by Random purposive sampling as many as 16 samples with symptoms of viral diseases from 16 villages in Langkat Regency. The genomic DNA was isolated from the samples and analyzed the virus using specific primers. The indications of infected rice leaf showed dwarf plants, yellowing leaves, few plant saplings, dark spots, rust spots, and midrib and short leaf blades in all samples at 16 villages at Langkat Regency. The similarity analysis of the 16 samples based on symptoms analyzed by the Unwight Pair Group Method with Arithmetic Average (UPGMA) is displayed in the form of a dendogram. All collections of symptomatic rice leaves from 16 villages were clustered at 79% similarity based on symptoms of viral disease. The results of the PCR were successfully amplified and positively infected with Rice Tungro Bacilliform Virus (RTBV) indicated by the presence of a 430 bp band fragment after being analyzed by PCR (Polymerase Chain Reaction) technique.

First Report of Tobacco Streak Virus in Cannabis sativa in New York.

In 2022, virus-like symptoms were observed in a field of diverse hemp (Cannabis sativa L.) germplasm in Ontario County, New York. Less than 1% of plants exhibited stunting and curled leaves (Figure S1), consistent with tobacco streak virus (TSV) symptoms on other plants (Liu et al. 2022). Most typically, the plants were considerably reduced in overall size, with upwards, adaxial curling along the leaf margin with newer leaves appearing to be the most affected. Fifteen symptomatic plants representing nine accessions were tested for 12 viruses and viroids through Agdia Testing Services (Elkhart, IN). Of these, eight plants representing five accessions including: G 33204 21UO SD ('Cherry Wine S1'), G 33211 21UO SD ('Wife'), G 33225 22CL01 CL ('Candida #2'), G 33270 22UO SD ('Falkowski CBD Mix'), and G 33365 22UO SD ('Queen Dream'), were positive for TSV, a type of Ilarvirus in the Bromoviridae family. Presence of TSV was confirmed through enzyme-linked immunosorbent assay testing. TSV is a positive-sense, single-stranded RNA virus with a wide host range that can be transmitted by thrips, mechanical injury, seed, and pollen (Zambrana-Echevarría et al. 2021). To confirm the presence of TSV, two putatively TSV-infected samples were subjected to RNA-Seq analysis. RNA was extracted using the RNeasy Plant Mini Kit (Qiagen, Aarhus, Denmark) per manufacturer's direction. Stranded RNA libraries were prepared using the Illumina TruSeq Stranded Total RNA with Ribo-Zero Plant kit (San Diego, California, USA). Paired-end 2x150bp sequencing was performed on an Illumina NovaSeq6000 sequencer. RNA-Seq data was trimmed using the fastp program (Chen et al. 2018) with default parameters to remove adapter sequences and low-quality bases. After filtering, 49,696,041 and 56,126,804 paired-end reads were retained from 'Wife' and 'Falkowski CBD Mix' samples, respectively. Filtered RNA-seq reads were mapped to TSV genome accession GCF_000865505.1 using the bowtie2 (Langmead & Salzberg 2012) aligner with default parameters. From 'Wife' and 'Falkowski CBD Mix' samples, 153 and 139 reads mapped to the TSV reference genome. To further validate the presence of TSV reads, RNA-Seq data was analyzed using the Kraken2 pipeline (Wood et al. 2019). Using the Kraken2 virus database, reads associated with TSV (NCBI taxonomy ID: 12317) were identified. This analysis identified 172 and 151 TSV reads from 'Wife' and 'Falkowski CBD Mix,' respectively. Higher numbers of reads identified using the Kraken2 analysis is due to the more permissive k-mer matching approach implemented in Kraken2. Furthermore, we identified several other virus taxa in the samples. Of note, both samples had a high number of reads associated with Amazon lily mild mottle virus with 254,493 and 116,150 reads from 'Wife' and 'Falkowski CBD Mix,' respectively. Among other virus species belonging to Ilarviruses, Cassava Ivorian bacilliform virus and Cowpea chlorotic mottle viruses were detected from both samples. To further validate infection by TSV, samples from both ELISA-positive and ELISA-negative plants were subjected to PCR using the primers and protocol described in Zambrana-Echevarría et al. 2021. Amplification of an approximately 700 base-pair product was observed in the putatively ELISA-positive samples, but not in the ELISA-negative samples. The amplicons were further cloned into the pGEM-T Easy vector (Promega, Madison, WI, U.S.A) using the manufacturer's protocol and sequenced using M13 forward and M13 reverse primers (Functional Biosciences, Madison, WI, U.S.A). Sequencing results indicated considerable similarity to TSV genomes available in GenBank, between 88% and 99%. Raw sequence data generated from this study was deposited in NCBI under the bioproject ID PRJNA1009441. Though it cannot be ruled out that the observed symptoms were caused exclusively by TSV infection due to the high number of other viral reads, the results contribute to the literature that indicates hemp can host TSV and should be considered a potential source of TSV inoculum (Chiginsky et al. 2021). This new inoculum source could cause significant crop damage and economic loss when grown with TSV susceptible row and specialty crops.

Identification of a sugarcane bacilliform virus promoter that is activated by drought stress in plants

Sugarcane (Saccharum spp.) is an important sugar and biofuel crop in the world. It is frequently subjected to drought stress, thus causing considerable economic losses. Transgenic technology is an effective breeding approach to improve sugarcane tolerance to drought using drought-inducible promoter(s) to activate drought-resistance gene(s). In this study, six different promoters were cloned from sugarcane bacilliform virus (SCBV) genotypes exhibiting high genetic diversity. In β-glucuronidase (GUS) assays, expression of one of these promoters (PSCBV-YZ2060) is similar to the one driven by the CaMV 35S promoter and >90% higher compared to the other cloned promoters and Ubi1. Three SCBV promoters (PSCBV-YZ2060, PSCBV-TX, and PSCBV-CHN2) function as drought-induced promoters in transgenic Arabidopsis plants. In Arabidopsis, GUS activity driven by promoter PSCBV-YZ2060 is also upregulated by abscisic acid (ABA) and is 2.2–5.5-fold higher when compared to the same activity of two plant native promoters (PScRD29A from sugarcane and PAtRD29A from Arabidopsis). Mutation analysis revealed that a putative promoter region 1 (PPR1) and two ABA response elements (ABREs) are required in promoter PSCBV-YZ2060 to confer drought stress response and ABA induction. Yeast one-hybrid and electrophoretic mobility shift assays uncovered that transcription factors ScbZIP72 from sugarcane and AREB1 from Arabidopsis bind with two ABREs of promoter PSCBV-YZ2060. After ABA treatment or drought stress, the expression levels of endogenous ScbZIP72 and heterologous GUS are significantly increased in PSCBV-YZ2060:GUS transgenic sugarcane plants. Consequently, promoter PSCBV-YZ2060 is a possible alternative promoter for genetic engineering of drought-resistant transgenic crops such as sugarcane.

The P2 nucleic acid binding protein of Sugarcane bacilliform virus is a viral pathogenic factor.

Saccharum spp. is the primary source of sugar and plays a significant role in global renewable bioenergy. Sugarcane bacilliform virus (SCBV) is one of the most important viruses infecting sugarcane, causing severe yield losses and quality degradation. It is of great significance to reveal the pathogenesis of SCBV and resistance breeding. However, little is known about the viral virulence factors or RNA silencing suppressors and the molecular mechanism of pathogenesis. To systematically investigate the functions of the unknown protein P2 encoded by SCBV ORF2. Phylogenetic analysis was implemented to infer the evolutionary relationship between the P2 of SCBV and other badnaviruses. The precise subcellular localization of P2 was verified in the transient infiltrated Nicotiana benthamiana epidermal mesophyll cells and protoplasts using the Laser scanning confocal microscope (LSCM). The post-transcriptional gene silencing (PTGS) and transcriptional gene silencing (TGS) RNA silencing suppressor activity of P2 was analyzed, respectively. Furthermore, restriction digestion and RT-qPCR assays were conducted to verify the probable mechanism of P2 on repressing DNA methylation. To explore the pathogenicity of P2, a potato virus X-based viral vector was used to heterologously express SCBV P2 and the consequent H2O2 accumulation was detected by the 3,3'-diaminobenzidine (DAB) staining method. Phylogenetic analysis shows that SCBV has no obvious sequence similarity and low genetic relatedness to Badnavirus and Tungrovirus representatives. LSCM studies show that P2 is localized in both the cytoplasm and nucleus. Moreover, P2 is shown to be a suppressor of PTGS and TGS, which can not only repress ssRNA-induced gene silencing but also disrupt the host RNA-directed DNA methylation (RdDM) pathway. In addition, P2 can trigger an oxidative burst and cause typical hypersensitive-like response (HLR) necrosis in systemic leaves of N. benthamiana when expressed by PVX. Overall, our results laid a foundation for deciphering the molecular mechanism of SCBV pathogenesis and made progress for resistance breeding.

Performance analysis of various deep learning models for detecting rice diseases

<p>A major portion of the world’s population relies on rice as a staple diet, hence rice is essential to maintaining food security worldwide. Unfortunately, rice crops are susceptible to a number of illnesses that, if detected and treated promptly, can result in significant output losses. Expert visual inspection is a time-consuming and arbitrary part of the conventional procedures for diagnosing diseases in rice. An effective method for automated illness diagnosis in agriculture has evolved in recent years: deep learning, a branch of artificial intelligence. The objective of this research is to compare AlexNet, DCNN, MobileNet, GoogleNet, VGG16, ResNet50 and Xception, these are various deep learning models in order to choose the one that would produce the highest levels of accuracy, precision recall, specificity, and F1-score for detecting rice diseases. In this study we train the model for nine different types of rice diseases named as Rice Blast (Pyricularia oryzae), Rice Sheath Blight (Rhizoctonia Solani), Bacterial Leaf Blight (Xanthomonas oryzae pv. oryzae), Tungro Disease, Rice Grassy Stunt Virus (RGSV), Rice Yellow Mottle Virus (RYMV), Bakanae Disease (Fusarium moniliforme), Brown Spot (Cochliobolus miyabeanus) and Rice Tungro Bacilliform Virus (RTBV) with 30,000 images. For this we used the secondary dataset for analyzing the performance of models. We trained the model for both normalized and non-normalized dataset. After comparing the various models we get the better result from ResNet50 model with accuracy of 97.50%.</p>

Harnessing Viral Promoters for Targeted RNAi: Engineering Phloem-Specific Resistance Against Tomato Yellow Leaf Curl Thailand Virus.

Phloem-specific promoter efficiently triggers graft-transmissible RNA interference (gtRNAi). We leveraged a phloem-specific promoter derived from the Rice tungro bacilliform virus, optimizing the RNAi mechanism's efficiency and specificity. Here, we detail the construction of phloem-specific promoter-based gtRNAi system and its application through grafting experiments, demonstrating its effectiveness in inducing tomato yellow leaf curl Thailand virus (TYLCHTV) resistance in non-transgenic scions. This strategy presents a practical application for protecting crops against viruses without genetically modifying the entire plant.

P4 protein of an Indian isolate of rice tungro bacilliform virus modulates gene silencing.

Plant hosts and their viral pathogens are engaged in a constant cycle of defense and counter-defense as part of a molecular arms race, principal among them being the plant RNAi defense and the viral RNAi suppressor counter-defense. Rice tungro bacilliform virus (RTBV), member of the family Caulimoviridae, genus Tungrovirus, species Tungrovirus oryzae, infects rice in South- and Southeast Asia and causes severe symptoms of stunting, yellow-orange discoloration and twisting of leaf tips. To better understand the possible counter-defensive roles of RTBV against the host RNAi defense system, we explored the ability of the P4 protein of an Indian isolate of RTBV to act as a possible modulator of RNAi. Using a transient silencing and silencing suppression assay in Nicotiana benthamiana, we show that P4 not only displays an RNAi suppressor function, but also potentially enhances RNAi. The results also suggests that the N-terminal 168 amino acid residues of P4 are sufficient to maintain RNAi suppressor activity. Taken together with the earlier reports this work strengthens the view that the P4 protein carries out RNAi suppressor and a potential RNAi enhancer function.

Phylogenetic analysis of endogenous viral elements in the rice genome reveals local chromosomal evolution in Oryza AA-genome species.

Rice genomes contain endogenous viral elements homologous to rice tungro bacilliform virus (RTBV) from the pararetrovirus family Caulimoviridae. These viral elements, known as endogenous RTBV-like sequences (eRTBVLs), comprise five subfamilies, eRTBVL-A, -B, -C, -D, and -X. Four subfamilies (A, B, C, and X) are present to a limited degree in the genomes of the Asian cultivated rice Oryza sativa (spp. japonica and indica) and the closely related wild species Oryza rufipogon. The eRTBVL-D sequences are widely distributed within these and other Oryza AA-genome species. Fifteen eRTBVL-D segments identified in the japonica (Nipponbare) genome occur mostly at orthologous chromosomal positions in other AA-genome species. The eRTBVL-D sequences were inserted into the genomes just before speciation of the AA-genome species. Ten eRTBVL-D segments are located at six loci, which were used for our evolutionary analyses during the speciation of the AA-genome species. The degree of genetic differentiation varied among the eRTBVL-D segments. Of the six loci, three showed phylogenetic trees consistent with the standard speciation pattern (SSP) of the AA-genome species (Type A), and the other three represented phylogenies different from the SSP (Type B). The atypical phylogenetic trees for the Type B loci revealed chromosome region-specific evolution among the AA-genome species that is associated with phylogenetic incongruences: complex genome rearrangements between eRTBVL-D segments, an introgression between the distant species, and low genetic diversity of a shared eRTBVL-D segment. Using eRTBVL-D as an indicator, this study revealed the phylogenetic incongruence of local chromosomal regions with different topologies that developed during speciation.

Identification of resistance to Sugarcane mosaic virus, Sugarcane streak mosaic virus, and Sugarcane bacilliform virus in new elite sugarcane accessions in India.

Sugarcane mosaic and leaf fleck diseases are significant viral diseases affecting sugarcane crops in India. The use of resistant sugarcane varieties is considered the most economical and effective approach to manage viral diseases, especially in vegetatively propagated crops such as sugarcane. Sugarcane mosaic virus (SCMV) and Sugarcane streak mosaic virus (SCSMV) are the primary pathogens responsible for mosaic disease in sugarcane-growing regions of India. Sugarcane bacilliform virus (SCBV), causing leaf fleck disease, is also often found in mixed infections with mosaic symptoms. The study aimed to identify new sources of resistance by screening sugarcane germplasm for resistance to SCMV, SCSMV, and SCBV. The screening was carried out under high inoculum using the infector row method in both plant and ratoon crops. Out of 129 genotypes tested, only 8 were found to be free of mosaic viruses, indicating a rare occurrence of resistant sources. The study revealed that mosaic disease is widespread, with nearly 95% of tested varieties/genotypes being infected with mosaic viruses. SCMV, SCSMV, and SCBV were detected in 121 out of 129 genotypes using the RT-PCR and PCR assays. Based on their response to the viruses, the tested genotypes were categorized into different resistance grades: highly resistant (grade 1), resistant (grade 2), moderately resistant (grade 3), susceptible (grade 4), and highly susceptible (grade 5). The results of the study provide valuable information about elite resistance resources that can be used for the prevention and control of mosaic disease. These resistant genotypes could also serve as potential donors for mosaic and leaf fleck disease resistance in breeding programs.

Sugarcane bacilliform viruses in Ethiopia: genetic diversity and transmission by pink sugarcane mealybug

Sugarcane bacilliform viruses in Ethiopia: genetic diversity and transmission by pink sugarcane mealybug

First Report of Cotton Leafroll Dwarf Virus infection of Malvaviscus arboreus in China.

Cotton leafroll dwarf virus (CLRDV, genus Polerovirus, family Solemoviridae) has been reported to infect cotton in Brazil, Argentina, India, Thailand and Timor-Leste (Agrofoglio YC et al. 2017; Corrêa RL et al. 2005; Mukherjee et al. 2012; Ray et al. 2016; Sharman et al. 2015), and in the United States (Ali and Mokhtari et al. 2020; Avelar et al. 2019). It has also been recently reported to infect Cicer arietinum (chickpea) in Uzbekistan and Hibiscus syriacus in Korea (Igori et al. 2022; Kumari et al. 2020). In China, the natural infection of plants by CLRDV has not been reported previously. In August 2017, leaf samples were collected from a wild plant of Malvaviscus arboreus (Malvaceae) exhibiting symptoms including leaf yellowing and distorting in Tengchong County of Yunnan Province. Leaves were used for total RNA extraction using TRIzol Reagent (Invitrogen, USA). Small RNA library construction and deep sequencing was performed on illumina HiSeqTM 2000 platform by Novogene Bioinformatic Technology Co., Ltd (Beijing, China). A total of 11, 525, 708 raw reads were obtained and further computationally analyzed by Perl scripts. The adaptors were removed and the obtained 7, 520, 902 clean reads with size of 18- to 26-nucleotide (nt) were aligned with the GenBank virus RefSeq database using Bowtie software. These reads were mainly mapped to the genomes of hibiscus bacilliform virus (genusBadnavirus,familyCaulimoviridae), hibiscus chlorotic ringspot virus (genus Betacarmovirus, family Procedovirinae), hibiscus latent Singapore virus (genus Tobamovirus, family Virgaviridae) and CLRDV isolate ARG (accession no. GU167940). The average coverage depth of clean reads mapped to CLRDV genome was 97.76%. Contigs greater than 50 nt were used to search for similar sequences by BLASTx, and 107 contigs were annotated as homologous to CLRDV isolates. To confirm CLRDV infection, reverse transcription polymerase chain reaction (RT-PCR) was performed using the specific primer pair CLRDV-F (5'-TCCACAGGAAGTATCACGTTCG-3') and CLRDV-R (5'-CCTTGTGTGGTTTGATTCGTGA-3') designed based on two of these contigs well-aligned to the genome of CLRDV isolate ARG. An amplicon of 1095-bp size was amplified, and was sequenced by Sanger sequencing (TsingKe Biological Technology, Chengdu, China), and BLASTn search results showed a maximum nucleotide identity of 95.45% with CLRDV isolate CN-S5, an isolate obtained from soybean aphid host in China (accession no. KX588248). To obtain more information on this CLRDV isolate, four primer pairs were designed and used for RT-PCR amplification (TableS1). The amplicons with size of about 860-, 1400-, 3200- and 1100-bp, were obtained separately and assembled into a complete genome sequence up to 5, 865-nt in length (isolate YN, deposited under GenBank accession no. MN057665). BLASTn showed the highest nucleotide similarity of 94.61% with CLRDV isolate CN-S5. From 2018 to 2022, additional M. arboreus samples with leaf yellowing or curling symptoms (9 from Shapingba District in Chongqing City, 5 from Nanchong City in Sichuan Province, 9 from Kunming City and 12 from Tengchong County in Yunnan Province) were collected and tested for CLRDV by RT-PCR using primer pairs CLRDV-F/CLRDV-R. The nucleotide sequences of the CLRDV P0 gene in two samples from Tengchong County were obtained by Sanger sequencing and deposited under GenBank (CLRDV isolate TCSL1 P0 gene, accession no. OQ749809; CLRDV isolate TCSW2 P0 gene, accession no. OQ749809). To our knowledge, this is the first report of CLRDV naturally infecting Malvaviscus arboreus in China, thus extending the information on its geographical distribution and host range. Malvaviscus arboreus is a widely cultivated ornamental plant in Yunnan Province, China. The natural occurrence of CLRDV not only affects the ornamental value of Malvaviscus arboreus, but also poses a potential threat to cotton production in China. This study will assist further surveillance of CLRDV infection and future development of effective protection strategies against CLRDV in China.

Detection of Sugarcane bacilliform virus in diseased sugarcane plants in Andhra Pradesh, India by serological and molecular methods.

Sugarcane leaf fleck incited by Sugarcane bacilliform virus is emerging as a major disease and affecting exchange of sugarcane germplasm and cultivation worldwide. Roving surveys conducted in 162 fields belonging to 81 villages spread over 14 sugarcane growing districts of Andhra Pradesh during 2021-2022 revealed 8 to 44% incidence of the disease. Mean maximum fleck disease incidence was reported in Anakapalli district (33.00%) followed by Srikakulam district (22.66%), whereas least incidence was observed in Alluri Sitharamaraju district (9.33%). The early and sensitive detection of pathogens is vital and necessary to reduce the danger of introducing new diseases or pathogen strains into sugarcane growing regions. Both serological and molecular methods were used in proposed investigation to identify the virus at the protein and nucleic acid levels. DAS-ELISA results were positive for 50 suspected SCBV infected sugarcane leaf samples out of 81, with mean absorbance (A405) values ranging from 0.50 to 2.20. Further PCR assays were performed using SCBV-specific primers targeting RT/RNase H coding region which is frequently employed as a taxonomic marker for species delineation in Badnaviruses. Out of 81 symptomatic samples collected, 61 samples gave positive results, and no amplification was observed in healthy control and negative control. Results made it evident that PCR was more sensitive than DAS-ELISA. Low virus concentration or variation in virus strains may be the reason for the low detection rate in DAS-ELISA in the current study. Extensive roving surveys conducted for the incidence of leaf fleck disease for the first time in the state of Andhra Pradesh revealed severe occurrence of leaf fleck disease under field conditions.

Bioecology and Management of Major Insect Pests of Rice in West Bengal

West Bengal is considered a large producer state of rice in India with an output of nearly 55.48% of total crop production of Bengal. In West Bengal, rice has grown in three different cropping seasons such as Aus (autumn rice), Aman (winter rice) and Boro (summer rice), contributing about 20.69% to the total net State Domestic Product (SDP). Rice production in West Bengal hampered by many biotic stresses mostly by stem borers [Scirphophaga incertulas (Walker, 1863), Sesamia inference (Walker, 1856), Chilo polychrysus (Meyrick, 1932), Chilo suppressalis(Meyrick, 1863)] leaf folder [Cnaphalocrocis medinalis (Guenee, 1854)], plant hoppers [Nilaparvata lugens(Stal, 1854), Sogatella furcifera (Horvath, 1899)], leaf hoppers [Nephotettix virescens (Distant, 1908), Nephotettix nigropictus (Stal, 1870)], rice hispa [Dicladispa armigera (Olivier, 1808)], rice bugs [Leptocorisa acuta (Thunberg, 1783), Leptocorisa oratorius (Fabricius, 1794), Brevennia rehi (Lindinger, 1943)], gall midge [Orseolia oryzae (Wood-Mason, 1889)], etc. Demographic parameter has shown that rice pests are greatly affecting rice host either caused directly by creating dead heart (DH), hopperburn, onion leaf, silver shoot during different stages of harvesting or by indirectly transmitting grassy stunt virus (RGSV), rice ragged stunt virus (RRSV), rice tungro viruses (RTV), rice tungro bacilliform virus (RTBV) etc. resulting 9.5% production loss. For minimize the infestation farmers use different rice varieties which provide resistance effect in pest population. For controlling rice pest population farmers mostly use chemical insecticide [e.g. fipronil, acephate, fipronil, monocrotophos] which exhibit good results against rice pests. Beside those other techniques including using biological and biochemical pesticides [neem oil, karanj oil, mahua oil, conidial suspension of M. anisopliae], pheromones [(Z)-11-hexadecinal, (Z)-9-hexadecenal], allomones [5: 1 mixture of 2-(E)-octenyl acetate and octanol 3-octenal], cultutal methods [synchronized planting, using of trap crop etc.] are effective for controlling rice pests. The population ecology based sustainable management of such pest species will support E 3 strategy [Ecosystem service-based ecological engineering for ecological pest management (ESS-EE-EPM)] of pest management for successful cultivation of rice in the near future.

Endogenous like sequence Rice Tungro Bacilliform Virus (RTBV) fragments in the local rice genome and its role in controlling tungro disease

Tungro disease is one of the important diseases in rice plants because it can cause a large decrease in production. Therefore, tungro disease needs to be controlled in wise ways that ensure environmental sustainability. This can be done by using resistant varieties, while also preventing rice from resistance to the green leafhopper vector Nepothettix virescens, the death of natural enemies, resurgence and emergence of secondary pests which become the main pests. The territory of Indonesia has many very diverse rice germ plasm which its resistance properties can be used to control tungro disease. Resistance genes are an effective control machine especially when combined with other control strategies. One of the triggers for the resistance of rice varieties is eRTBV, which is a group of RTBV nucleotides in rice DNA. The purpose of this study was to detect the presence of the eRTBV piece in the rice genome and its role in controlling tungro disease. The method used wasthe insilico method by comparing the eRTBV found in local rice varieties with that found in Genbank. The results indicated that endogenous RTBV was joined into the rice genome at 1200 bp using the primer eRTBV-7. Nucleotide blast results showed that the similarity of endogenous Indonesian local rice RTBV to endogenous RTBV in the genbank ranged between 95% and 99%, then protein blast results showed that the eRTBV fragment of local rice varieties contained ORF 3 RTBV protein with a range of similarity to the genbank of 66.9% and 93.35%. The role of eRTBV is to produce the Isoleucine protein motif which produces a physical, chemical and biochemical resistance system capable of inhibiting the green leafhopper virus vector (Nepothettix virescens) and the growth and development of tungro disease, RTBV.

Salibu Rice Cultivation in Tungro Endemis Region

Salibu is modified ratoon rice which has advantages in economic and time-saving. Tungro disease is one of probable handicap for salibu to be controlled in their endemic area. Rice virus tungro disease is caused by Rice Tungro Bacilliform Virus (RTBV) and Rice Tungro Spherical Virus (RTSV) which are transmitted by leafhopper. The research goals are to know the growth of leafhopper population and the spread of the tungro disease in salibu and conventional rice farming system as well as their effect on yield. The research was conducted in split plot experiment and designed in a randomized block design. The main plot were 1) salibu system and 2) conventional system (non-salibu). The subplots were varieties 1) TN1 (sensitive variety), 2) Ciherang (commonly used by the farmer), and 3) Inpari 36 (tungro new resistant variety). The results revealed that the tungro attack rate, dry milled grain yield, and 1000 dry grain weight were significantly higher in conventional cultivation than salibu (P < 0.05). The number of leafhopper populations tended to be higher in salibu system than in conventional system. The number of natural enemies did not show a particular pattern related to green leafhopper's presence. The Shannon Wiener diversity index for natural enemies ranged from low to medium. Regarding to the results, salibu system is not recommended yet in the tungro endemic areas. Keywords: Salibu, rice tungro virus disease, green leafhopper, natural enemies

Molecular detection of rice tungro bacilliform virus (RTBV) on weed host in South Sulawesi

Rice tungro bacilliform Virus (RTBV) is one of the viruses that cause tungro disease in rice plants causing leaf yellow and plant stunting. Some weed species have also been described as potential hosts for RTBV in addition to rice. However, RTBV cannot be diagnosed in weeds only by looking at their morphological symptoms because weeds infected with RTBV do not show typical symptoms. Hence this research was conducted to detect the presence of RTBV molecularly on several weed hosts in rice fields in South Sulawesi. The polymerase Chain Reaction technique with coat protein gene-specific primers was used to detect RTBV. A total of 29 leaf samples consisting of 12 weed species collected from Sidrap, Pinrang, Maros, and Gowa were then extracted genomic DNA from the leaf samples for PCR analysis. The results of PCR analysis showed that weed samples that tested positive for RTBV were Panicum repens (Lawawoi/Sidrap), Echinochloa crusgalli (Lawawoi/Sidrap), Cyperus difformis (Lanrang/Sidrap), Echinochloa crusgalli (Palangga/Gowa), Fimbristilys miliacea (Lanrang/Sidrap), and Eleusine indica (Lawawoi/Sidrap) which were indicated by the presence of RTBV virus DNA fragments with 430 bp in length in all these weeds. Weeds infected by RTBV formed 2 clusters. The first cluster consisted of RTBV in P.repens and E.crusgalli from Lawawoi/Sidrap, RTBV in C.difformis and F.miliacea from Lanrang/Sidrap, and RTBV in E.indica from Palangga/Gowa. At the same time, the second cluster consisted of RTBV in E.indica from Lawawoi/Sidrap.

Candidate miRNAs from Oryza sativa for Silencing the Rice Tungro Viruses

Rice tungro disease (RTD), caused by Rice tungro bacilliform virus (RTBV) and Rice tungro spherical virus (RTSV) is one of the most prominent viral diseases in Asian countries. This virus disease problem seems to have been accentuated in those countries by causing a series of outbreaks over the years after being first reported in International Rice Research Institute (IRRI), Philippines, in 1963. One of the effective ways to combat viruses is through RNA silencing. microRNA is an important player in the RNA silencing mechanism. Genome sequences analysis shows RTBV-SP isolate (8 Kb) is composed of four open reading frames (ORF 1, ORF 2, ORF 3, and ORF 4), meanwhile, RTSV-SP (12 Kb) consists of one open reading frame encoded by seven different polyproteins (P1, CP1, CP2, CP3, NTP, Pro, and Rep). Therefore, this study investigated possible rice-encoded miRNAs targeted on RTBV and RTSV using in silico analysis. Five bioinformatics tools were employed using five different prediction algorithms: miRanda, RNA22, RNAhybrid, Tapirhybrid, and psRNATarget. The results revealed each RTBV and RTSV can be silenced by three potentially best candidate rice-encoded miRNA. For RTBV, osa-miR5510 (accession no. MIMAT0022143), osa-miR3980a-3p (accession no. MIMAT0019676), and osa-miR3980b-3p (accession no. MIMAT0019678) are being predicted by all five algorithms. Meanwhile, for RTSV, three miRNAs predicted are osa-miR414 (accession no. MIMAT0001330), osa-miR5505 (accession no. MIMAT00221138) and osa-miR167a-3p (accession no. MIMAT0006780). The predicted data provide useful material for developing RTBV and RTSV-resistant rice varieties.

Silencing suppressor protein PRT of rice tungro bacilliform virus interacts with the plant RNA silencing-related protein SGS3

BackgroundRice tungro bacilliform virus (RTBV) is a double stranded DNA containing virus which causes the devastating tungro disease of rice in association with an RNA virus, rice tungro spherical virus. RNA silencing is an evolutionarily conserved antiviral defence pathway in plants as well as in several classes of higher organisms. Many viruses, in turn, encode proteins which are termed Viral Suppressor of RNA Silencing (VSR) because they downregulate or suppress RNA silencing. ResultsUsing an RNA silencing suppressor assay we show that RTBV protease (PRT) acts as a mild VSR. A truncated version of PRT gene abolished the silencing suppression activity. We also show in planta interaction of PRT with the SGS3 protein of Solanum tuberosum and Arabidopsis thaliana using bimolecular fluorescence complementation assay (BIFC). Transient expression of PRT in Nicotiana benthamiana caused an increased accumulation of the begomovirus Sri Lankan cassava mosaic virus (SLCMV) DNA-A, which indicated a virulence function imparted on an unrelated virus. ConclusionThe finding supports the idea that PRT acts as suppressor of RNA silencing and this action may be mediated by its interaction with SGS3.