Healthy Tobacco Plants Research Articles

First report of Pseudopithomyces palmicola causing leaf spot on tobacco in China.

In July 2023, a new leaf spot disease emerged on tobacco leaves in Meitan County, Guizhou Province, China (27°20'18" - 28°12'30"N, 107°15'36" - 107°41'08"E, average altitude 972 meters). Initially, the symptoms showed raised yellow-brown spots; subsequently, the lesions expanded and became broken and perforated, leading to a significant loss of economic value, the prevalence rate exceeded 30%. For isolation, two tissue fragments (0.2 × 0.2 cm) of symptomatic leaves were sterilized in 75% ethanol for 30 s, 3% NaClO for 2 min, and were washed 3 times in sterilized distilled water, and were subsequently inoculated on potato dextrose agar (PDA), and incubated at 28°C for 9 days in the dark. The two strains CW16 and CW28 were isolated using the single hyphae method (Nouri et al. 2023). Both strains formed pale to yellow white colonies on PDA. Conidia had three constricted transverse septa and 1 to 2 longitudinal septa in the central cells, with thick and hyaline conidiophores and mostly globose, pale brown conidia with slightly constricted septa, their average size were measured as 13.4-22.4×8.358-13.347 μm (n = 50). Genomic DNA was extracted from the isolated strains CW16 and CW28. The internal transcribed spacer regions 1 and 2 as well as 5.8S nuclear ribosomal RNA (ITS), large subunit nrRNA (LSU), and partial DNA-directed RNA polymerase II second largest subunit (RPB2) genes were amplified using primers (Cui et al. 2023). The sequences had been deposited in GenBank under accession numbers ITS: PP024201, PP024205; LSU: PP024207, PP024209; RPB2: PP060480, PP060481. The sequences analysis revealed a high similarity of 99.74 to 100% between strains CW16 and CW28 with P. palmicola isolate KM42 (ITS OQ875842, LSU OQ875844, RPB2 OQ883943) in GenBank. Using BLAST for homology matching, two isolates (CW16, CW28) and with the sequences of the ten type isolates from GenBank, phylogenetic analysis was conducted using the Maximum Likelihood method in MEGA (11.0) software based on ITS, LSU and RPB2 sequences, which showed that strains CW16, CW28 clustered in the same score as the Pseudopithomyces palmicola, confirming the morphological and molecular characteristics identification. The pathogenicity tests were conducted on healthy tobacco plants with 4-5 leaves (Fig. S1B), the isolated strains, CW16 and CW28, were used to inoculate the healthy tobacco leaves, while blank PDA was used as a control. All plants were maintained in a greenhouse at 28°C with a relative humidity of 90%. After 9 days, necrotic spots were observed on all tobacco leaves inoculated with CW16 and CW28 fungal plugs, while the blank PDA-inoculated tobacco leaves showed no symptoms. Based on morphological and molecular characteristics, the same pathogen P. palmicola was identified from the inoculated leaves, fulfilling Koch's postulates. This study represents the first reported of tobacco leaf spot caused by P. palmicola in China and provides a theoretical basis for future prevention and control measures.

Impact of soil sterilization on antagonistic efficiency against tobacco mosaic virus and the rhizosphere bacterial community in Nicotiana benthamiana

Soil microorganisms play a critical role in influencing plant growth and managing soil pathogens. Tobacco mosaic virus (TMV) induces significant economic losses in global agriculture and can impact the composition and function of soil microbial communities. Despite its importance, the interactions between viruses and soil microbial communities remain inadequately understood. In this study, we employed 16S rRNA gene sequencing coupled with bioinformatics analyses to thoroughly investigate the bacterial communities and physicochemical properties of the rhizosphere soils of healthy tobacco plants under both sterilized (WJ) and non-sterilized (YJ) conditions, as well as TMV-infected tobacco plants under both sterilized (WT) and non-sterilized (YT) conditions. Our findings demonstrated that TMV infection significantly modifies the physicochemical properties and bacterial community structure of rhizosphere soils, with these changes being more pronounced in non-sterilized soils. Moreover, the YT samples exhibited a more intricate network of bacterial interactions. They showed significant differences from WT samples in key bacterial genera that might be involved in the response to or antagonism of TMV. The genera Burkholderia-Caballeronia-Paraburkholderia and Dyella were highlighted. These results suggest that rhizosphere microorganisms actively respond to TMV infection, with a more pronounced response observed in non-sterilized soils. This study provides novel insights into the microbial dynamics associated with TMV infection and underscores the importance of soil microbial communities in plant health and disease resistance. Additionally, it offers an experimental framework for future research on soil-borne diseases, emphasizing the pivotal role of soil microbiota in disease ecology and soil impact.

Bacterial wilt affects the structure and assembly of microbial communities along the soil-root continuum

BackgroundBeneficial root-associated microbiomes play crucial roles in enhancing plant growth and suppressing pathogenic threats, and their application for defending against pathogens has garnered increasing attention. Nonetheless, the dynamics of microbiome assembly and defense mechanisms during pathogen invasion remain largely unknown. In this study, we aimed to investigate the diversity and assembly of microbial communities within four niches (bulk soils, rhizosphere, rhizoplane, and endosphere) under the influence of the bacterial plant pathogen Ralstonia solanacearum.ResultsOur results revealed that healthy tobacco plants exhibited more diverse community compositions and more robust co-occurrence networks in root-associated niches compared to diseased tobacco plants. Stochastic processes (dispersal limitation and drift), rather than determinism, dominated the assembly processes, with a higher impact of drift observed in diseased plants than in healthy ones. Furthermore, during the invasion of R. solanacearum, the abundance of Fusarium genera, a known potential pathogen of Fusarium wilt, significantly increased in diseased plants. Moreover, the response strategies of the microbiomes to pathogens in diseased and healthy plants diverged. Diseased microbiomes recruited beneficial microbial taxa, such as Streptomyces and Bacilli, to mount defenses against pathogens, with an increased presence of microbial taxa negatively correlated with the pathogen. Conversely, the potential defense strategies varied across niches in healthy plants, with significant enrichments of functional genes related to biofilm formation in the rhizoplane and antibiotic biosynthesis in the endosphere.ConclusionOur study revealed the varied community composition and assembly mechanism of microbial communities between healthy and diseased tobacco plants along the soil-root continuum, providing new insights into niche-specific defense mechanisms against pathogen invasions. These findings may underscore the potential utilization of different functional prebiotics to enhance plants’ ability to fend off pathogens.

First Report of Target Spot Caused by Rhizoctonia solani AG-5 on Tobacco in China.

Tobacco is one of the vital economic crops in China. Nevertheless, tobacco diseases cause substantial economic losses each year. Tobacco target spot is a fungal disease which commonly found on the leaves. While both sexual and asexual reproduction can occur, asexual reproduction is much more common in tobacco. In June 2022, target spot was found on tobacco leaf samples from Yibin, Sichuan Province and Meitan, Guizhou Province, China. The typical symptoms were light brown tissue with concentric ring marks, and the necrotic part of the disease spot was fragile and forming perforation after falling off. The diseased tissue in the sample was cut off and sterilized in 75% ethanol for 1 min, and rinsed three times in sterilized distilled water. Finally, the tissues were placed on potato glucose agar (PDA) medium with kanamycin (0.1 mg/mL). After incubation at 28 °C in darkness for 3 days,the culture of the isolate grew in the form of radial mycelium on PDA dishes, the mycelium was white initially, turned brown generally at the later stage, and finally thickened and separated with the growth of the culture. Nine pathogenic strains were isolated, including four isolates from Yibin and five from Meitan. They were all used for pathogen identification. Genomic DNA of each isolate was extracted using the CATB method, and PCR analysis was performed with primers specifically designed to detect individual fusion groups or fusion subgroups of solani: AG-1 IA, IB, and IC; AG-3 PT; AG-4 HG-I, HG-II and HG-III; AGs-5-6 and P-21-22. Among the 11 specific primer pairs, only AG-5-specific primer amplified the fungal DNA, indicating that the nine isolates tested all belonged to the R. solani AG-5 fusion group. BLASTn search was performed on the gene sequences obtained from these strains and they deposited in GenBank under accession no. OP647851-OP647859. These gene sequences were aligned with the voucher specimen R. solani AG-5, with more than 99% similarity . The nine isolates were then tested for mycelial anastomosis reactions using the R. solani AG-5 standard strain following the method described by Ogoshi (1987). A decrease in the diameter of the mycelia at the anastomosis site and death of adjacent cells were observed, indicating their anastomosis response. Therefore, these nine strains were identified as R. solani AG-5 based on morphological and genetic analysis. Subsequently, one pathogenic strain from Meitan and another one from Yibin were selected for pathogenicity verification. Mycelial PDA blocks (6 mm in diameter) of the two isolates were inoculated on healthy tobacco plants, while leaves containing only PDA blocks were used as controls. A total of 6 replicates were conducted. After inoculation, they were incubated at 85% relative humidity and 15 to 25 °C. Koch's hypothesis was confirmed by reisolating pathogens from diseased leaves 5 days after inoculation. Typical symptoms were observed on tobacco plants inoculated with the pathogen strains but not on control tobacco plants. To the best of our knowledge, tobacco target spot has been reported caused by R. solani AG-3, AG-6 and AG-2.1 groups in the field in China and in Argentina. Up until now, this is the first report of R. solani AG-5 causing tobacco target spot on tobacco in the field in China. It was also found to be highly virulent to chickpea in Turkey. Due to serious damages caused by this disease in the last five years in China, more attention should be paid in disease control measures to avoid economic losses. In addition, it also provides some theoretical help for the damage caused by this pathogen on other hosts and helps people to better understand Rhizoctonia solani AG-5.

Leptosphaerulina americana causing holing disease on tobacco in China.

Leptosphaerulina americana causing holing disease on tobacco in China.

Physiochemical Properties and Microflora of the Rhizosphere Soil of Tobacco Plants with and without Bacterial Wilt

Bacterial wilt is a destructive soilborne disease caused by Ralstonia solanacearum, posing a severe threat to plants in the Solanaceae family. It impacts on tobacco productivity worldwide. This study was conducted to analyze the changes in the soil’s physical and chemical properties, the number of microbes, and the bacterial diversity of the rhizosphere soil before and after the wilt disease. The rhizosphere soil of healthy and diseased tobacco plants was collected from Pucheng, Nanping, Fujian Province, Southern China. The results revealed significant differences in the trends of physical and chemical properties of the soil of healthy and diseased plants. The soil pH, available potassium (K), available phosphorous (P), and organic matter contents (SOM) were lower in the rhizosphere soil for healthy plants than for pre-diseased plants (HW). Only the available P, among all physical and chemical properties in the rhizosphere of diseased plants (HS), was significantly lower than those for pre-diseased plants (HW), changing from 149.59 mg/kg to 59.19 mg/kg. The order of numbers of the three main microbes in the rhizosphere soil for healthy plants (HC) and pre-diseased plants was the following: bacteria > actinomycetes > fungi. The number of actinomycetes in the soil of the diseased tobacco plants increased significantly. A comparison of the rhizosphere soil of diseased and healthy tobacco plants showed that the relative abundance of the bacterial community in the rhizosphere soil of the pathogenic tobacco plants changed significantly. The community diversity was increased, and the Pseudomonadaceae, to which the bacterial pathogen of bacterial wilt belonged, rose to a certain extent. Both pre-diseased and healthy plants showed changes in the physical and chemical properties, microbial quantity, and microbial diversity, thus proving that tobacco disease was closely related to the soil’s ecological environment.

Potential risk of plant viruses entering disease cycle in surface water in protected vegetable growing areas of Eastern China.

With the expansion of protected vegetable growing areas (PVGAs), viral plant diseases have become more prevalent, causing severe economic losses to the vegetable production industry in China. At present, researches on plant viruses mainly focus on plants, but there is only a few reports on the species of viruses in surface water from PVGAs. The surface water samples in PVGAs are representative to a certain extent, which has an important reference value for studying the characteristics of plant viruses in surface water. The purpose of this study was to identify the diversity and the possibility of entering disease infection cycle of plant viruses in water samples collected from PVGAs in eastern China. A total of 144 water samples were collected, and eight plant viruses including tobacco mosaic virus (TMV, 8.33%), cucumber green mottle mosaic virus (CGMMV, 33.33%), pepper mild mottle virus (PMMoV, 6.94%), cucumber mosaic virus (CMV, 0.69%), tomato masaic virus (ToMV, 3.47%), tomato mottle mosaic virus (ToMMV, 0.69%), tomato chlorosis virus (ToCV, 4.17%), and tomato yellow leaf curl virus (TYLCV, 5.56%) were examined using RT-PCR and PCR. The species of viruses in surface water varied greatly by location. CGMMV, TMV, ToCV, ToMV, ToMMV, and TYLCV were identified in Shandong, a northern part of Eastern China, whereas only PMMoV was found in Shanghai, a southern part of Eastern China. After healthy tobacco plants were inoculated with the concentrated solutions of TMV, ToMV, CGMMV, and PMMoV, could cause disease in healthy tobacco, indicating that the plant viruses in the concentrated solution have the infectivity, and the plant viruses in surface water have the possibility of entering the infection cycle of disease. The results will improve the understanding of the potential risks of waterborne disease transmission.

Characterization of the belowground microbial community and co-occurrence networks of tobacco plants infected with bacterial wilt disease.

Characterizing the microbial communities associated with soil-borne disease incidence is a key approach in understanding the potential role of microbes in protecting crops from pathogens. In this study, we compared the soil properties and microbial composition of the rhizosphere soil and roots of healthy and bacterial wilt-infected tobacco plants to assess their potential influence on plant health. Our results revealed that the relative abundance of pathogens was higher in diseased plants than in healthy plants. Moreover, compared with healthy plants, there was a significantly higher microbial alpha diversity in the roots and rhizosphere soil of diseased plants. In addition, we detected a lower abundance of certain plant microbiota, including species in the genera Penicillium, Trichoderma, and Burkholderia in the rhizosphere of diseased plants, which were found to be significantly negatively associated with the relative abundance of Ralstonia. Indeed, compared with healthy plants, the co-occurrence networks of diseased plants included a larger number of associations linked to plant health. Furthermore, structural equation modeling revealed that these specific microbes were correlated with disease suppression, thereby implying that they may play important roles in maintaining plant health. In conclusion, our findings provide important insights into the relationships between soil-borne disease incidence and changes in the belowground microbial community. These findings will serve as a basis for further research investigating the use of specific plant-associated genera to inhibit soil-borne diseases.

Microbial Cross-Talk: Dissecting the Core Microbiota Associated With Flue-Cured Tobacco (Nicotiana tabacum) Plants Under Healthy and Diseased State.

Bacterial wilt caused by Ralstonia solanacearum is a devastating disease of flue-cured tobacco production which poses significant yield losses all around the world. In this study, we evaluated the rhizosphere microbiome of healthy and bacterial wilt-infected (diseased) flue-cured tobacco plants through amplification of V3-V4 and ITS1-5f variable regions of 16S and internal transcribed spacer (ITS) rRNA. The study was based on the location (Qujing, Shilin, and Wenshan), plant components (rhizosphere soil and roots), and sample types (healthy and diseased) to assess the diversity of bacterial and fungal communities. Bacterial and fungal communities present in roots primarily emanated from rhizosphere soil. Healthy flue-cured tobacco plants exhibit high microbial diversity compared to diseased plants. Among three variables, plant components significantly influence the diversity of microbial communities, whereas rhizosphere soil harbors higher microbial diversity than roots. Bacterial phyla Cyanobacteria and Proteobacteria were found in high relative abundance in roots and rhizosphere soil samples, respectively. As far as fungi is concerned, a high relative abundance of Ascomycota and Basidiomycota was found in both rhizosphere soil and root. Bacterial genera such as Bacillus, Bradyrhizobium, Ensifer, Neorhizobium, and Lysobacter related to plant growth promotion and disease suppressing abilities were dominant than fungal genera. Analysis of relative abundance at specie-level revealed that most fungal species are pathogenic to flue-cured tobacco and could provide a conducive environment for wilt infection. In conclusion, R. solanacearum significantly influences the microbial diversity of flue-cured tobacco plants and negatively affects the bacterial community composition. Altogether, our study demonstrates the complexity of bacterial and fungal communities that possibly interact with each other (microbe–microbe) and host (host–microbe). This cross-talk could be helpful for healthy flue-cured tobacco plant growth and to induce resistance against bacterial wilt disease.

Bacillus amyloliquefaciens WS-10 as a potential plant growth-promoter and biocontrol agent for bacterial wilt disease of flue-cured tobacco

BackgroundBacterial wilt disease caused by the soilborne bacterium Ralstonia solanacearum is a serious threat to flue-cured tobacco production. In this study, an indigenous disease suppressive Bacillus strain was isolated from the rhizosphere soil of healthy tobacco plants, and its biocontrol and plant growth promoting (PGP) potential were evaluated in in-vivo and in-vitro assays.ResultsThrough isolation and screening of 250 isolates, WS-10 was found to be the best candidate antagonistic strain against R. solanacearum (WS-001). In-vitro assays revealed that the isolated strain WS-10 (Bacillus amyloliquefaciens) showed an effective antagonistic activity against R. solanacearum WS-001 and several plant-pathogenic fungi. As promising PGP rhizobacteria, WS-10 had the ability of nitrogen fixation, solubilization of inorganic potassium and phosphate, and biosynthesis of indole-3-acetic. In a co-culture assay, it significantly inhibits the growth of WS-001. Our greenhouse experiments showed that the soil physicochemical properties and accumulation of dry matter contents in different plant parts (roots, stems, and leaves) were significantly increased in the presence of B. amyloliquefaciens WS-10. The soil treated with B. amyloliquefaciens WS-10 displayed significantly higher values of the average well color development index, the utilization ability of 6 types of carbon sources by rhizosphere microorganisms, and the diversity indices of the rhizosphere microbial communities. In planta assay, B. amyloliquefaciens WS-10 significantly reduced tobacco bacterial wilt disease incidence by up to 73.36, 43.82, and 86.82% under three different treatments by improving the functional diversity and biological activity of the soil microbial community.ConclusionsObtained findings suggested that B. amyloliquefaciens WS-10 had an excellent potential as a growth-promoting and biocontrol agent of tobacco bacterial wilt disease due to its multiple beneficial traits of nutrient solubilization and disease suppression. Thus, we conclude that B. amyloliquefaciens WS-10 was a high potential PGP and biocontrol strain for healthy production of tobacco crop.

Effects of Meloidogyne incognita on the fungal community in tobacco rhizosphere

The often widespread and serious Root-Knot Nematode (RKN) disease is an important soil-borne disease affecting tobacco production. This study aimed to understand micro-ecological changes caused by RKN disease and interactions between disease and rhizosphere soil fungal communities. The 18S rRNA gene sequencing was used to study changes in rhizosphere fungal community of tobacco plants having RKN disease. In June 2018, a paired comparison was performed between rhizosphere fungal community structures of healthy tobacco plants and those with RKN disease [...]

Genomic insights on fighting bacterial wilt by a novel Bacillus amyloliquefaciens strain Cas02.

SummaryBacterial wilt, caused by the Ralstonia solanacearum, can infect several economically important crops. However, the management strategies available to control this disease are limited. Plant growth‐promoting rhizobacteria (PGPR) have been considered promising biocontrol agents. In this study, Bacillus amyloliquefaciens strain Cas02 was isolated from the rhizosphere soil of healthy tobacco plants and evaluated for its effect on plant growth promotion and bacterial wilt suppression. Strain Cas02 exhibited several growth‐promoting–related features including siderophore production, cellulase activity, protease activity, ammonia production and catalase activity. Moreover, strain Cas02 showed a significant inhibitory growth effect on R. solanacearum, and its active substances were separated and identified to be macrolactin A and macrolactin W by HPLC‐DAD‐ESI‐MS/MS. Both greenhouse and field experiments demonstrated a good performance of Cas02 in plant growth promotion and bacterial wilt suppression. To explore the underlying genetic mechanisms, complete genome sequencing was performed and the gene clusters responsible for antibacterial metabolites expression were identified. Overall, these findings suggest that the strain Cas02 could be a potential biocontrol agent in bacterial wilt management and a source of antimicrobial compounds for further exploitation.

Whole Genome Sequence of Bacillus velezensis Strain GUMT319: A Potential Biocontrol Agent Against Tobacco Black Shank Disease.

Phytophthora nicotianae causes black shank, a serious soil-borne disease, in tobacco. In this study, the Bacillus strain GUMT319 was isolated from the rhizosphere of healthy tobacco plants grown in a field in Guizhou with a high incidence of tobacco black shank. Genome sequencing revealed that GUMT319 contained a single circular chromosome 3,940,023 bp in length, with 4,053 predicted genes and an average GC content of 46.6%. Based on phylogenomic analyses, GUMT319 was designated as Bacillus velezensis. The genome of GUMT319 contained more than 60 genes and 13 gene clusters that have previously been found to be active in antifungal mechanisms, biofilm formation, and chemotaxis motility. Additionally, confocal laser scanning microscopy and scanning electron microscopy showed that GUMT319 formed a spatially organized biofilm in vivo. In addition, lauric acid negatively regulated biofilm formation. This is the first study to report that nicotine in tobacco root exudates was a chemoattractant for biocontrol Bacillus strains. In this study, we identified new interactions between beneficial microorganisms and tobacco roots in the rhizosphere. Moreover, dual culture tests in vitro showed that GUMT319 inhibited the growth of P. nicotianae and also displayed inhibitory effects against eight other plant pathogens, namely, Colletotrichum scovillei, Colletotrichum capsici, Fusarium carminascens, Sclerotinia sclerotiorum, Alternaria alternata, Phomopsis sp., Phyllosticta sorghina, and Exserohilum turcicum. Furthermore, GUMT319 exhibited > 70% control efficiency against tobacco black shank in field experiments conducted in 2018–2020. Thus, GUMT319 was more effective in controlling the incidence of tobacco black shank than other treatments including fungicide application. Overall, these results suggested that GUMT319 (B. velezensis) could be used as a potential biocontrol agent against tobacco black shank.

Early detection of tomato spotted wilt virus infection in tobacco using the hyperspectral imaging technique and machine learning algorithms

The hyperspectral imaging technique was used for the non-destructive detection of tomato spotted wilt virus (TSWV) infection in tobacco at an early stage. Spectra ranging from 400 to 1000 nm with 128 bands from inoculated and healthy tobacco plants were analyzed by using three wavelength selection methods (successive projections algorithm (SPA), boosted regression tree (BRT), and genetic algorithm (GA)), and four machine learning (ML) techniques (boosted regression tree (BRT), support vector machine (SVM), random forest (RF), and classification and regression tress (CART)). The results indicated that the models built by the BRT algorithm using the wavelengths selected by SPA as the input variables obtained the best outcome for the 10-fold cross-validation with the mean overall accuracy of 85.2% and area under receiver operating curve (AUC) of 0.932. The band selection results and variable contribution analysis in BRT modeling jointly showed that the near-infrared (NIR) spectral region is informative and important for the differentiation of infected and healthy tobacco leaves. Different stages of post-inoculation were split according to the molecular identification and visual observation. The classification results at different stages indicated that the hyperspectral imaging data combined with ML methods and wavelength selection algorithms can be used for the early detection of TSWV in tobacco, both at the presymptomatic stage and during the period before the systematic infection can be detected by the molecular identification approach.

First report of tobacco root rot caused by Phytophthora nicotianae in Mexico

During 2014 and 2015, symptoms of root rot were observed in tobacco fields located in Veracruz, Mexico. Phytophthora colonies developing rosaceous pattern were consistently isolated on potato dextrose agar (PDA) and V8 juice agar media. Sporangia were papillate, noncaducous, measuring 40 to 49 x 33 to 37 μm, and with short pedicels. The sporangial shape was ovoid or ellipsoid. Chlamydospores were mostly solitary, intercalary, or terminal, measuring 29-33 μm in diameter. The oomycete was identified as Phytophthora nicotianae based on descriptions of Gallegly and Hong (2008). To confirm the identification, genomic DNA from a representative isolate was extracted, and the internal transcribed spacer (ITS) region was amplified by PCR and sequenced using the primers ITS5 and ITS4 (White et al., 1990). The resulting 896 bp sequence was deposited as GenBank Accession No. KR906059. BLASTn analysis showed a 97% similarity with those of P. nicotianae (GenBank Accession No. AB688370). Pathogenicity tests were performed twice on 20 healthy tobacco plants by immersion of roots in a zoospore suspension adjusted to 1 x 104 zoospores ml-1. Ten tobacco plants inoculated with sterile distilled water served as controls. Inoculated plants developed root rot symptoms after 10 days, whereas no symptoms were observed on the control plants. The oomycete present on inoculated plants was morphologically identical to that originally observed on diseased plants, thus fulfilling Koch’s postulates. To our knowledge, this is the first report of tobacco root rot caused by Phytophthora nicotianae in Mexico.

Potato virus Y: Contact Transmission, Stability, Inactivation, and Infection Sources.

In glasshouse experiments, two isolates of Potato virus Y 'O' strain (PVYO) were transmitted from infected to healthy potato plants by direct contact when leaves were rubbed against each other, when cut surfaces of infected tubers were rubbed onto leaves, and to a limited extent, when blades contaminated with infective sap were used to cut healthy potato tubers. However, no tuber-to-tuber transmission occurred when blades were used to cut healthy tubers after cutting infected tubers. When leaf sap from potato plants infected with two PVYO isolates was kept at room temperature, it was highly infective for 6 to 7 h and remained infectious for up to 28 h. Also, when sap from infected leaves with one isolate was applied to five surfaces (cotton, hessian, metal, rubber vehicle tire, and wood) and left to dry for up to 24 h before each surface was rubbed onto healthy tobacco plants, PVYO remained infective for 24 h on tire and metal, 6 h on cotton and hessian, and 3 h on wood. The effectiveness of disinfectants at inactivating this isolate was evaluated by adding them to sap from infected leaves which was then rubbed onto healthy tobacco plants. None of the plants became infected when bleach (42 g/liter sodium hypochlorite, diluted 1:4) or Virkon-S (potassium peroxymonosulfate 50% wt/wt, diluted to 1%) was used. A trace of infection remained after using nonfat milk powder (20% wt/vol). PVY infection sources were studied in 2011-2012 in the main potato growing regions of southwest Australia. In tests on >17,000 potato leaf samples, PVY was detected at low levels in seed (4/155) and ware (6/51) crops. It was also detected in volunteer potatoes from a site with a previous history of PVY infection in a seed crop. None of the 15 weed species tested were PVY infected. Plants of Solanum nigrum were symptomlessly infected with PVYO after sap inoculation, and no seed transmission was detected (>2,500 seeds). This study demonstrates PVYO can be transmitted by contact and highlights the need to include removal of volunteer potatoes and other on-farm hygiene practices (decontaminating tools, machinery, clothing, etc.) in integrated disease management strategies for PVY in potato crops.

A previously undescribed potyvirus isolated and characterized from arborescent Brugmansia

A suspected virus disease was identified from an arborescent Brugmansia x candida Pers. (syn. Datura candida Pers.) tree. The causal agent was aphid transmissible at low rates. Viral particles were purified from infected tobacco tissue, analyzed, and purified virions were inoculated into healthy tobacco plants to recreate the symptoms. The virions had a mean length of 720-729 nm, and infected cells contained inclusion bodies typical of potyvirus infections. Analysis of infected tissues and purified virions with a panel of potyvirus-specific antibodies confirmed identification as a potyvirus. Viral host range, dilution end point, thermal tolerance and aphid transmission characteristics were examined. The viral genome (9761 nt) is typical of potyviruses, with the closest related potyvirus being pepper mottle virus, at 72 % nt sequence identity. Based on conventions for naming novel potyviruses, the virus was determined to be a member of a previously undescribed species, tentatively named "Brugmansia mosaic virus" (BruMV).

First Report of Tomato spotted wilt virus on Pepper in Montenegro.

In April 2009, chlorotic and necrotic ring spots, chlorotic line patterns, and stunting were observed on greenhouse-grown pepper plants in the vicinity of Podgorica, Montenegro. Disease symptom incidence was estimated at 40%. Symptomatic leaves were tested for the presence of Tomato spotted wilt virus (TSWV) with a commercial double-antibody sandwich (DAS)-ELISA diagnostic kit (Bioreba AG, Reinach, Switzerland). Commercial positive and negative controls were included in each ELISA. TSWV was detected serologically in 33 of 75 pepper samples. The virus was mechanically transmitted from ELISA-positive pepper samples to Nicotiana tabacum cv. Samsun using chilled 0.05 M phosphate buffer (pH 7) containing 0.1% sodium sulfite (1). Inoculated test plants produced chlorotic and necrotic concentric rings and necrotic spots, consistent with symptoms caused by TSWV on N. tabacum. For further confirmation of TSWV infection, reverse transcription (RT)-PCR was performed with the One-Step RT-PCR Kit (Qiagen, Hilden, Germany) using three sets of primers: S70-for/S890-rev (2) and S574-for/S1433-rev (3), both specific to the nonstructural (NSs) gene; and S1983-for/S2767-rev (2), specific to the nucleocapsid protein (N) gene. Total RNAs from naturally infected pepper and symptomatic N. tabacum cv. Samsun plants were extracted with the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany). Total RNAs obtained from the Italian isolate of TSWV (GenBank Accession No. DQ398945) and healthy tobacco plants were used as positive and negative controls, respectively. The expected sizes of the RT-PCR products (820, 859, and 784 bp) were amplified from symptomatic pepper samples but not from healthy tissues. The PCR product obtained from isolate Is-344 using primers specific to N gene was purified by a QIAquick PCR Purification Kit (Qiagen), cloned into the pGEM-T Easy Vector (Promega, Madison, WI) and sequenced in both directions using the same primer pair as in RT-PCR. The sequences amplified with the two primer pairs specific to the NSs gene were obtained by direct sequencing (Bio-Fab Research Srl, Pomezia, Italy) and joined using MEGA4 software. Sequence analysis of the complete N gene (777 bp; GenBank Accession No. GU369717) revealed that the TSWV isolate originating from Montenegro shared 98.2 to 99.7% nucleotide identity (98.1 to 100% amino acid identities) with corresponding TSWV sequences deposited in GenBank. The Montenegrin isolate Is-344 was most closely related to Italian isolates from tomato (GU369725) and eggplant (GU369720). The partial (1,257 bp) nucleotide sequence of NSs gene (GU369737) showed 96 to 99.8% nucleotide identity (96.9 to 100% amino acid identity) with previously reported TSWV sequences, and in this case the highest identity was with French isolates from tomato (FR692835) and lettuce (FR692831). To our knowledge, this is the first report on the occurrence of TSWV in Montenegro. Data of this study sheds light on the importance of further survey studies and inspections of TSWV-susceptible crops cultivated in Montenegro.

An invasive whitefly feeding on a virus-infected plant increased its egg production and realized fecundity.

BackgroundPlant-pathogenic begomoviruses have a complex association with their insect vectors. The interactions of begomoviruses and reproduction of their vectors are poorly understood. Bemisia tabaci is known to transmit many begomoviruses, and the spread of B. tabaci, especially the B and Q ‘biotypes’, has been accompanied by the epidemics of begomoviruses. One of these identified disease-causing agents was Tomato yellow leaf curl China virus (TYLCCNV).Methodology/Principal FindingsIn this study, we compared the egg production and realized fecundity of two ‘biotypes’ or putative species of the whitefly B. tabaci, including the alien invasive B and the indigenous ZHJ1 from Zhejiang, China, feeding on either healthy or TYLCCNV-infected tobacco plants. The ovary of the whitefly was composed of 12–22 telotrophic ovarioles. According to the morphology of the oocytes and level of yolk content, oocytes in ovarioles were divided into four developmental phases (I-IV). Significantly higher proportion of immature oocytes (phase II, III) and mature oocytes (phase IV) was observed in ovary of females that fed on TYLCCNV-infected tobacco compared to that on healthy plants. Moreover, there was significant increase of eggs laid of B whitefly that fed on TYLCCNV-infected tobacco plants during the early developmental stages. In contrast, the proportion of oocytes of different developmental phases and eggs laid had no significant differences between ZHJ1 whiteflies feeding on TYLCCNV-infected and non-infected host plants.Conclusions/SignificanceThe invasive B whitefly benefits from feeding on a begomovirus-infected plant through increased egg production and realized fecundity.

Vector-virus mutualism accelerates population increase of an invasive whitefly.

The relationships between plant viruses, their herbivore vectors and host plants can be beneficial, neutral, or antagonistic, depending on the species involved. This variation in relationships may affect the process of biological invasion and the displacement of indigenous species by invaders when the invasive and indigenous organisms occur with niche overlap but differ in the interactions. The notorious invasive B biotype of the whitefly complex Bemisia tabaci entered China in the late 1990s and is now the predominant or only biotype in many regions of the country. Tobacco curly shoot virus (TbCSV) and Tomato yellow leaf curl China virus (TYLCCNV) are two whitefly-transmitted begomoviruses that have become widespread recently in south China. We compared the performance of the invasive B and indigenous ZHJ1 whitefly biotypes on healthy, TbCSV-infected and TYLCCNV-infected tobacco plants. Compared to its performance on healthy plants, the invasive B biotype increased its fecundity and longevity by 12 and 6 fold when feeding on TbCSV-infected plants, and by 18 and 7 fold when feeding on TYLCCNV-infected plants. Population density of the B biotype on TbCSV- and TYLCCNV-infected plants reached 2 and 13 times that on healthy plants respectively in 56 days. In contrast, the indigenous ZHJ1 performed similarly on healthy and virus-infected plants. Virus-infection status of the whiteflies per se of both biotypes showed limited effects on performance of vectors on cotton, a nonhost plant of the viruses. The indirect mutualism between the B biotype whitefly and these viruses via their host plants, and the apparent lack of such mutualism for the indigenous whitefly, may contribute to the ability of the B whitefly biotype to invade, the displacement of indigenous whiteflies, and the disease pandemics of the viruses associated with this vector.