Plant Pathogenic Viruses Research Articles

Exogenous RNAs for Gene Regulation and Plant Resistance.

Recent investigations documented that plants can uptake and process externally applied double-stranded RNAs (dsRNAs), hairpin RNAs (hpRNAs), and small interfering RNAs (siRNAs) designed to silence important genes of plant pathogenic viruses, fungi, or insects. The exogenously applied RNAs spread locally and systemically, move into the pathogens, and induce RNA interference-mediated plant pathogen resistance. Recent findings also provided examples of plant transgene and endogene post-transcriptional down-regulation by complementary dsRNAs or siRNAs applied onto the plant surfaces. Understanding the plant perception and processing of exogenous RNAs could result in the development of novel biotechnological approaches for crop protection. This review summarizes and discusses the emerging studies reporting on exogenous RNA applications for down-regulation of essential fungal and insect genes, targeting of plant viruses, or suppression of plant transgenes and endogenes for increased resistance and changed phenotypes. We also analyze the current understanding of dsRNA uptake mechanisms and dsRNA stability in plant environments.

Involvement of sweet pepper CaLOX2 in jasmonate-dependent induced defence against Western flower thrips.

Insect herbivory can seriously hinder plant performance and reduce crop yield. Thrips are minute cell‐content‐feeding insects that are important vectors of viral plant pathogens, and are serious crop pests. We investigated the role of a sweet pepper (Capsicum annuum) lipoxygenase gene, CaLOX2, in the defense of pepper plants against Western flower thrips (Frankliniella occidentalis). This was done through a combination of in‐silico, transcriptional, behavioral and chemical analyses. Our data show that CaLOX2 is involved in jasmonic acid (JA) biosynthesis and mediates plant resistance. Expression of the JA‐related marker genes, CaLOX2 and CaPIN II, was induced by thrips feeding. Silencing of CaLOX2 in pepper plants through virus‐induced gene silencing (VIGS) resulted in low levels of CaLOX2 transcripts, as well as significant reduction in the accumulation of JA, and its derivatives, upon thrips feeding compared to control plants. CaLOX2‐silenced pepper plants exhibited enhanced susceptibility to thrips. This indicates that CaLOX2 mediates JA‐dependent signaling, resulting in defense against thrips. Furthermore, exogenous application of JA to pepper plants increased plant resistance to thrips, constrained thrips population development and made plants less attractive to thrips. Thus, a multidisciplinary approach shows that an intact lipoxygenase pathway mediates various components of sweet pepper defense against F. occidentalis.

Draft genome sequence of broad-spectrum antifungal-producing Bacillus velezensis C4341 isolated from a saline-alkali soil sample in China.

Bacterial micro-organisms from the genus Bacillus have a broad spectrum of antimicrobial activity against plant pathogenic bacteria, fungi and viruses. Bacillus velezensis has been extensively used as a biocontrol agent in the agricultural field. In the present study, B. velezensis strain C4341 was isolated from saline-alkali soil in Xinjiang, China, producing a wide range of antifungal compounds of different structures. The whole genome of B. velezensis C4341 was sequenced using an Illumina HiSeq™ 2000 sequencing system, was assembled using SOAPdenovo and was optimised with GapCloser. The genome size of strain C4341 was calculated at 3 963 750bp with a G+C content of 46.4%. The whole genome was assembled into 4019 open reading frames (ORFs), of which 5.9% and 1.6% were related to antagonistic secondary metabolites and antimicrobial resistance, respectively. The whole genome sequence of B. velezensis C4341 will be beneficial in comparative analysis of the genus Bacillus and shows future prospects in antifungal application for sustainable agriculture.

Proximal remote sensing to differentiate nonviruliferous and viruliferous insect vectors – proof of concept and importance of input data robustness

Proximal remote sensing is being widely studied as a noninvasive method to partially automate diagnostics of plants and insects. The hypothesis that proximal remote sensing can be used to differentiate specimens of adult beet leafhoppers (Circulifer tenellus) that were nonviruliferous or viruliferous for beet curly top virus (BCTV) was tested. A key aspect of applications of proximal remote sensing is the ‘robustness’ or repeatability of input reflectance data. Many factors may contribute to low input reflectance data robustness; these include: (i) issues related to the consistency of proximal remote sensing conditions (light intensity and spectral composition, ambient temperature), (ii) insect specimen preparation (projection angle, storage and handling), and (iii) insect specimen characteristics (age, growing conditions, variety/biotype, host plant). This study demonstrates that nonviruliferous and viruliferous specimens of adult beet leafhoppers possess unique body reflectance features and, therefore, can be differentiated. However, insect specimen preparation (removal of wings and placement) markedly affected the classification accuracy. Addition of experimental noise to input reflectance data was conducted to simulate varying degrees of input reflectance data robustness. The potential of developing reflectance‐based diagnostic tools for detection of plant pathogenic viruses in insects is discussed, with an emphasis on input data robustness.

Double-Stranded RNAs in Plant Protection Against Pathogenic Organisms and Viruses in Agriculture.

Recent studies have shown that plants are able to express the artificial genes responsible for the synthesis of double-stranded RNAs (dsRNAs) and hairpin double-stranded RNAs (hpRNAs), as well as uptake and process exogenous dsRNAs and hpRNAs to suppress the gene expression of plant pathogenic viruses, fungi, or insects. Both endogenous and exogenous dsRNAs are processed into small interfering RNAs (siRNAs) that can spread locally and systemically through the plant, enter pathogenic microorganisms, and induce RNA interference-mediated pathogen resistance in plants. There are numerous examples of the development of new biotechnological approaches to plant protection using transgenic plants and exogenous dsRNAs. This review summarizes new data on the use of transgenes and exogenous dsRNAs for the suppression of fungal and insect virulence genes, as well as viruses to increase the resistance of plants to these pathogens. We also analyzed the current ideas about the mechanisms of dsRNA processing and transport in plants.

Treating wheat seeds with neonicotinoid insecticides does not harm the rhizosphere microbial community.

Wheat aphids damage wheat plants directly by feeding on them and indirectly by transmitting plant pathogenic viruses, both of which result in low yield and plant death. Due to their high root absorption and systemic characteristics, neonicotinoid insecticidal seed treatments are increasingly applied to control wheat aphids throughout the growing season in China. Ecological concerns are raised in some research, because neonicotinoids can persist and accumulate in soils. They are prone to leach into waterways, and are found in crop nectars and pollens, where they may be harmful to pollinators. Less information is available about the effect of neonicotinoid seed treatments on soil microorganisms. Here, we posed the hypothesis that neonicotinoids are not harmful to soil microbial communities. We tested our hypothesis by evaluating the effects of two neonicotinoids, imidacloprid and clothianidin, on soil microbiomes using high-throughput sequencing during three points in the wheat growth season. Except for the imidacloprid-treated soil in the seedling stage, the community richness and diversity were not affected according to Chao1, ACE and the Shannon indices, and species distribution histogram at the phylum level. However, Beta diversity indices showed that the species richness of the bacterial and fungal community was suppressed by neonicotinoids in seedling stage (high neonicotinoids concentrations), whereas by the reviving period, the changes reverted into stimulation of the soil microorganisms (low neonicotinoids concentrations). Overall, the general microbiome recovered at the end of the wheat planting season. Generally, wheat seed dressing with neonicotinoid insecticides control aphids during the entire growth period, and have no lasting adverse effects on the soil microbiome. This study provides an understanding of the influence of neonicotinoids on crop land ecology at the level of soil microbe communities.

Design, Synthesis, and Biological Activity of Novel Myricetin Derivatives Containing Amide, Thioether, and 1,3,4-Thiadiazole Moieties.

A series of myricetin derivatives containing amide, thioether, and 1,3,4-thiadiazole moieties were designed and synthesized, and their antiviral and antibacterial activities were assessed. The bioassays showed that all the title compounds exhibited potent in vitro antibacterial activities against Xanthomonas citri (Xac), Ralstonia solanacearum (Rs), and Xanthomonas oryzae pv. Oryzae (Xoo). In particular, the compounds 5a, 5f, 5g, 5h, 5i, and 5l, with EC50 values of 11.5–27.3 μg/mL, showed potent antibacterial activity against Xac that was better than the commercial bactericides Bismerthiazol (34.7 μg/mL) and Thiodiazole copper (41.1% μg/mL). Moreover, the in vivo antiviral activities against tobacco mosaic virus (TMV) of the target compounds were also tested. Among these compounds, the curative, protection, and inactivation activities of 5g were 49.9, 52.9, and 73.3%, respectively, which were better than that of the commercial antiviral Ribavirin (40.6, 51.1, and 71.1%, respectively). This study demonstrates that myricetin derivatives bearing amide, thioether, and 1,3,4-thiadiazole moieties can serve as potential alternative templates for the development of novel, highly efficient inhibitors against plant pathogenic bacteria and viruses.

A Loop-mediated Isothermal Amplification (LAMP) Assay for Rapid Identification of <em>Bemisia tabaci</em>

The whitefly Bemisia tabaci (Gennadius) is an invasive pest of considerable importance, affecting the production of vegetable and ornamental crops in many countries around the world. Severe yield losses are caused by direct feeding, and even more importantly, also by the transmission of more than 100 harmful plant pathogenic viruses. As for other invasive pests, increased international trade facilitates the dispersal of B. tabaci to areas beyond its native range. Inspections of plant import products at points of entry such as seaports and airports are, therefore, seen as an important prevention measure. However, this last line of defense against pest invasions is only effective if rapid identification methods for suspicious insect specimens are readily available. Because the morphological differentiation between the regulated B. tabaci and close relatives without quarantine status is difficult for non-taxonomists, a rapid molecular identification assay based on the loop-mediated isothermal amplification (LAMP) technology has been developed. This publication reports the detailed protocol of the novel assay describing rapid DNA extraction, set-up of the LAMP reaction, as well as interpretation of its read-out, which allows identifying B. tabaci specimens within one hour. Compared to existing protocols for the detection of specific B. tabaci biotypes, the developed method targets the whole B. tabaci species complex in one assay. Moreover the assay is designed to be applied on-site by plant health inspectors with minimal laboratory training directly at points of entry. Thorough validation performed under laboratory and on-site conditions demonstrates that the reported LAMP assay is a rapid and reliable identification tool, improving the management of B. tabaci.

A Loop-mediated Isothermal Amplification (LAMP) Assay for Rapid Identification of Bemisia tabaci.

The whitefly Bemisia tabaci (Gennadius) is an invasive pest of considerable importance, affecting the production of vegetable and ornamental crops in many countries around the world. Severe yield losses are caused by direct feeding, and even more importantly, also by the transmission of more than 100 harmful plant pathogenic viruses. As for other invasive pests, increased international trade facilitates the dispersal of B. tabaci to areas beyond its native range. Inspections of plant import products at points of entry such as seaports and airports are, therefore, seen as an important prevention measure. However, this last line of defense against pest invasions is only effective if rapid identification methods for suspicious insect specimens are readily available. Because the morphological differentiation between the regulated B. tabaci and close relatives without quarantine status is difficult for non-taxonomists, a rapid molecular identification assay based on the loop-mediated isothermal amplification (LAMP) technology has been developed. This publication reports the detailed protocol of the novel assay describing rapid DNA extraction, set-up of the LAMP reaction, as well as interpretation of its read-out, which allows identifying B. tabaci specimens within one hour. Compared to existing protocols for the detection of specific B. tabaci biotypes, the developed method targets the whole B. tabaci species complex in one assay. Moreover the assay is designed to be applied on-site by plant health inspectors with minimal laboratory training directly at points of entry. Thorough validation performed under laboratory and on-site conditions demonstrates that the reported LAMP assay is a rapid and reliable identification tool, improving the management of B. tabaci.

Developing a Programmable, Self-Assembling Squash Leaf Curl China Virus (SLCCNV) Capsid Proteins into “Nanocargo”-like Architecture

A new era has begun in which pathogens have become useful scaffolds for nanotechnology applications. In this research/study, an attempt has been made to generate an empty cargo-like architecture from a plant pathogenic virus named Squash leaf curl China virus (SLCCNV). In this approach, SLCCNV coat protein monomers are obtained efficiently by using a yeast Pichia pastoris expression system. Further, dialysis of purified SLCCNV-CP monomers against various pH modified (5-10) disassembly and assembly buffers produced a self-assembled "Nanocargo"-like architecture, which also exhibited an ability to encapsulate magnetic nanoparticles in vitro. Bioinformatics tools were also utilized to predict the possible self-assembly kinetics and bioconjugation sites of coat protein monomers. Significantly, an in vitro biocompatibility study using SLCCNV-Nanocargo particles showed low toxicity to the cells, which eventually proved as a potential nanobiomaterial for biomedical applications.

Effects of humic acid and plant growth-promoting rhizobacteria (PGPR) on induced resistance of canola to Brevicoryne brassicae L.

The cabbage aphid, Brevicoryne brassicae L. (Hem: Aphididae), is an important pest of canola that can considerably limit profitable crop production either through direct feeding or via transmission of plant pathogenic viruses. One of the most effective approaches of pest control is the use of biostimulants. In this study, the effects of humic acid, plant growth-promoting rhizobacteria (PGPR), and integrated application of both compounds were investigated on life table parameters of B. brassicae, and the tolerance of canola to this pest. B. brassicae reared on plants treated with these compounds had the lower longevity, fecundity, and reproductive period compared with control treatment. The intrinsic rate of natural increase (r) and finite rate of increase (λ) were lowest on PGPR treatment (0.181 ± 0.004 day-1 and 1.198 ± 0.004 day-1, respectively) and highest on control (0.202 ± 0.005 day-1 and 1.224 ± 0.006 day-1, respectively). The net reproductive rate (R0) under treatments of humic acid, PGPR and humic acid + PGPR was lower than control. There was no significant difference in generation time (T) of B. brassicae among the tested treatments. In the tolerance test, plants treated with PGPR alone or in integrated with humic acid had the highest tolerance against B. brassicae. The highest values of total phenol, flavonoids, and glucosinolates were observed in treatments of PGPR and humic acid + PGPR. Basing on the antibiosis and tolerance analyses in this study, we concluded that canola plants treated with PGPR are more resistant to B. brassicae. These findings could be useful for integrated pest management of B. brassicae in canola fields.

Honey bee‐assisted surveillance for early plant virus detection

Incursions of plant viral pathogens are a primary concern for horticulture as they can significantly impact crop yields and require expensive management. Early detection of plant viruses is critical for effective plant biosecurity because it enables growers to respond quickly and limit their spread. However, current surveillance methods relying on enzyme‐linked immunosorbent assay and PCR testing of plant material can only operate at a limited scale. It quickly becomes unfeasible to test a large number of samples for area‐wide surveillance of multiple viruses and ineffective in novel virus discovery. Advances in high‐throughput sequencing (HTS) platforms have greatly improved our surveillance capability and its value demonstrated for screening plant material for viral pathogens. However, while HTS can improve detection, it does not necessarily address the issue of sampling effort needed to achieve early detection. The solution is to couple HTS with a method of constant surveillance and sampling from potentially infected plants. We propose that when managed honey bee (Apis mellifera) hives are brought to flowering crops to deliver essential pollination services, they can also provide valuable surveillance by effectively “sampling” entire crops as they collect pollen and nectar. Combining the ability of honey bees to create a representative sample from a large number of plants with the sensitivity of HTS offers a powerful system for early detection of plant viruses. We describe a novel approach to plant biosecurity where HTS virus detection in honey bees was found to provide earlier detection of plant virus incursions in Australia than current surveillance programmes. In 2013–2014, a national virus survey of honey bees was conducted, which used HTS screening of pooled honey bee samples across Australia. This HTS data simultaneously identified multiple plant viruses in these samples and showed the presence of cucumber green mottle mosaic virus in several states of Australia before its subsequent detection from diseased plant material.

Full sequence of the coat protein gene is required for the induction of pathogen-derived resistance against sugarcane mosaic virus in transgenic sugarcane.

Sugarcane mosaic virus (SCMV) is a plant pathogenic virus of the family Potyviridae that causes chlorosis, stunting and significantly reduced sugar productivity in sugarcane. Pathogen-derived resistance is a method used to develop SCMV-resistant sugarcane by overexpression of viral DNA. In this study, the gene encoding the coat protein (CP) of SCMV was amplified by reverse transcriptase PCR from symptomatic sugarcane leaves and used to generate transgenic sugarcane. Nucleotide sequence analysis of amplified cDNA indicated that the 998-bp-long cDNA, termed ScMVCp cDNA, codes for the CP of SCMV from the PS881 isolate. The ScMVCp cDNA was inserted into the binary vector pRI101-ON with two constructs, a full nucleotide sequence (p927) and a sequence coding for N-terminally truncated protein (p702). The constructs were then introduced into sugarcane using Agrobacterium-mediated transformation. Southern blot analysis showed a single hybridized DNA copy inserted into the genome of transgenic sugarcane lines. The inserted genes were expressed at both the RNA transcript and protein levels in the transgenic sugarcane. The highest expression was found in transgenic lines 10, 11 and 13 from the p927 construct. Artificial infection by the virus showed that p927 generated a higher resistance to virus compared with p702. This resistance was passed on to the second generation of transgenic sugarcane with 100 and 20-40% levels of resistance in the p927 and p702 transgenic lines, respectively. This report shows that the full sequence of the CP gene is required to disrupt viral assembly and packaging, thereby generating resistance to SCMV infection.

Biological control of plant pathogens by Bacillus species

Bacteria from the Bacillus group are microorganisms that inhabit a large number of different habitats. They are well known as producers of a wide array of antagonistic compounds of different structures, having between 5 to 8% of the total genome devoted to biosynthesis of secondary metabolites. Most important bioactive molecules from the genus Bacillus are non-ribosomally synthesized peptides and lipopeptides, polyketide compounds, bacteriocins and siderophores. Lipopeptides from Bacillus have very complex mechanisms of biosynthesis catalyzed by non-ribosomal peptide synthetases (NRPSs), large enzyme complexes with modular structure, with each module being in charge for the incorporation of a particular amino acid. In general, they have a broad spectrum of antagonistic activity against plant pathogenic bacteria, fungi and viruses. Most important molecules from this group, circular lipopeptides from surfactin, iturin and fengycin families affect the target cells on the membrane level. Bacillus strains exhibit their biocontrol capacity predominantly through inhibitory activity on the growth of plant pathogens, as well as inducing systemic resistance in plants and competing for ecological niches with plant pathogens. Our previous studies showed the presence of multiple biosynthetic operons for synthesis of non-ribosomal lipopeptides in the collection of natural isolates of Bacillus, with many strains having more than one of them. Several strains of Bacillus sp. that we have recently characterized showed very strong antibacterial and antifungal activity against phytopathogens. The PCR analysis showed the presence of biosynthetic operons for iturin, bacillomycin, fengycin and surfactin in tested strains. Measurement of the kinetics of production of antimicrobial substances showed that, in most cases, synthesis started at the beginning of exponential phase of growth, reaching the maximum of antimicrobial activity at the beginning of the stationary growth phase and stayed at this level for the whole duration of observed period. Preparations of cell-free supernatants of tested strains were active against many fungal and bacterial pathogens, in vitro and in vivo. Mass spectrometry and HPTLC bioautography analysis of purified compounds confirmed the presence of lipopeptides of mentioned families, hence confirming the biocontrol capacity of Bacillus isolates.

Predation of Aphis craccivora (Hemiptera: Aphididae) by Orius sauteri (Hemiptera: Anthocoridae) Under Different Temperatures

Aphids are important agricultural pests because of their feeding and, for many species, their ability to transmit plant pathogenic viruses. Orius sauteri (Poppius) (Hemiptera: Anthocoridae) is an important predator in northern China of many aphid species, including Aphis craccivora (Koch) (Hemiptera: Aphididae). We measured the functional response and intraspecific competition of O. sauteri under different densities of A. craccivora at several constant temperatures. Predation of O. sauteri on A. craccivora conformed to Holling's type II functional response at all temperatures tested. The predation capability of O. sauteri showed a significant correlation with temperature. The predation capacity of O. sauteri was highest at 25°C, at which temperature we found the highest instantaneous attack rate (a = 0.8711), the theoretical maximum number of aphids captured (181.36), and the shortest handling time (Th = 0.0055). Predation by O. sauteri increased progressively with temperature from 15 to 25°C, but then decreased at 30°C. Intraspecific competition of O. sauteri increased positively with predator density under all temperatures examined. Our results indicate O. sauteri has significant potential for use as a biocontrol agent, under a wide range of temperatures. The inverse density-dependent predation and intraspecific competition of O. sauteri suggests that to obtain the greatest degree of pest suppression, releases of O. sauteri should begin early in the cropping season and numbers released should take prey density into consideration.

Fabrication of virus metal hybrid nanomaterials: An ideal reference for bio semiconductor

Recently, Nanotechnology has made easier utilizing plant pathogens as a potential nanomaterial in biomedical applications. In this research work, we have exploited a devastating plant pathogenic virus of Squash leaf curl China virus (SLCCNV), as a nano-bio template (32 nm) to fabricate the gold and silver nanomaterials. This is achieved through the direct exposure of SLCCNV to gold chloride (HAuCl4) and silver nitrate (AgNO3) precursors at sunlight, resulted into SLCCNV-metallic-hybrid nanomaterials which are synthesized quick (∼5 min) and eco-friendly. However, virus hybrid nanomaterials are fabricated through the nucleation and growth of metal precursors over the pH-activated capsid of SLCCNV. Under the controlled fabrication process, it produced a highly arrayed virus-metallic-hybrid nanomaterial at nanoscale size limit. Its properties are thoroughly studied through spectroscopic techniques (UV–Vis, DLS, Raman) and electron microscopy (HRTEM & FESEM). In a follow-up study of cytotoxicity assay, the virus and its fabricated nanomaterials show better biocompatibility features even at high concentrations. Finally, the electrical conductivities of virus-metallic-hybrid nanomaterials (Au & Ag) are determined by simple “lab on a chip” system and Keithley's pico-ammeter. The result of electrical conductivity measurement revealed that hybrid nanomaterials have greater electrical conductive properties within the band-gap of semi-conductive materials. It is truly remarkable that a plant virus associated metal nanomaterials can be efficiently used as bio-semi-conductors which are the ideal one for biomedical applications.

Chloroplast Proteome of Nicotiana benthamiana Infected by Tomato Blistering Mosaic Virus.

Tymovirus is a genus of plant pathogenic viruses that infects several dicotyledonous plants worldwide, causing serious diseases in economically important crops. The known cytopathic effect on the host cell organelles involves chloroplast membrane deformation and the induction of vesicles in its periphery. These vesicles are known to be the location where tymoviral genomic RNA replication occurs. Tomato blistering mosaic virus (ToBMV) is a tymovirus recently identified in tomato plants in Brazil, which is able to infect several other plants, including tobacco. In this work, we investigated the chloroplast proteomic profile of ToBMV-infected N. benthamiana using bidimensional electrophoresis (2-DE) and mass spectrometry, aiming to study the virus-host interaction related to the virus replication and infection. A total of approximately 200 spots were resolved, out of which 36 were differentially abundant. Differential spots were identified by mass spectrometry including photosynthesis-related and defense proteins. We identified proteins that may be targets of a direct interaction with viral proteins, such as ATP synthase β subunit, RNA polymerase beta-subunit, 50S ribosomal protein L6 and Trigger factor-like protein. The identification of these candidate proteins gives support for future protein-protein interaction studies to confirm their roles in virus replication and disease development.

Study of the Metatranscriptome of Eight Social and Solitary Wild Bee Species Reveals Novel Viruses and Bee Parasites.

Bees are associated with a remarkable diversity of microorganisms, including unicellular parasites, bacteria, fungi, and viruses. The application of next-generation sequencing approaches enables the identification of this rich species composition as well as the discovery of previously unknown associations. Using high-throughput polyadenylated ribonucleic acid (RNA) sequencing, we investigated the metatranscriptome of eight wild bee species (Andrena cineraria, Andrena fulva, Andrena haemorrhoa, Bombus terrestris, Bombus cryptarum, Bombus pascuorum, Osmia bicornis, and Osmia cornuta) sampled from four different localities in Belgium. Across the RNA sequencing libraries, 88–99% of the taxonomically informative reads were of the host transcriptome. Four viruses with homology to insect pathogens were found including two RNA viruses (belonging to the families Iflaviridae and Tymoviridae that harbor already viruses of honey bees), a double stranded DNA virus (family Nudiviridae) and a single stranded DNA virus (family Parvoviridae). In addition, we found genomic sequences of 11 unclassified arthropod viruses (related to negeviruses, sobemoviruses, totiviruses, rhabdoviruses, and mononegaviruses), seven plant pathogenic viruses, and one fungal virus. Interestingly, nege-like viruses appear to be widespread, host-specific, and capable of attaining high copy numbers inside bees. Next to viruses, three novel parasite associations were discovered in wild bees, including Crithidia pragensis and a tubulinosematid and a neogregarine parasite. Yeasts of the genus Metschnikowia were identified in solitary bees. This study gives a glimpse of the microorganisms and viruses associated with social and solitary wild bees and demonstrates that their diversity exceeds by far the subset of species first discovered in honey bees.

SNARE-Encoding Genes VdSec22 and VdSso1 Mediate Protein Secretion Required for Full Virulence in Verticillium dahliae.

Proteins that mediate cellular and subcellular membrane fusion are key factors in vesicular trafficking in all eukaryotic cells, including the secretion and transport of plant pathogen virulence factors. In this study, we identified vesicle-fusion components that included 22 soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), four Sec1/Munc18 (SM) family proteins, and 10 Rab GTPases encoded in the genome of the vascular wilt pathogen Verticillium dahliae Vd991. Targeted deletion of two SNARE-encoding genes in V. dahliae, VdSec22 and VdSso1, significantly reduced virulence of both mutants on cotton, relative to the wild-type Vd991 strain. Comparative analyses of the secreted protein content (exoproteome) revealed that many enzymes involved in carbohydrate hydrolysis were regulated by VdSec22 or VdSso1. Consistent with a role of these enzymes in plant cell-wall degradation, pectin, cellulose, and xylan utilization were reduced in the VdSec22 or VdSso1 mutant strains along with a loss of exoproteome cytotoxic activity on cotton leaves. Comparisons with a pathogenicity-related exoproteome revealed that several known virulence factors were not regulated by VdSec22 or VdSso1, but some of the proteins regulated by VdSec22 or VdSso1 displayed different characteristics, including the lack of a typical signal peptide, suggesting that V. dahliae employs more than one secretory route to transport proteins to extracellular sites during infection.

Water-Mediated Transmission of Plant, Animal, and Human Viruses.

Viruses represent the most abundant and diverse of the biological entities in environmental waters, including the seas and probably also freshwater systems. They are important players in ecological networks in waters and influence global biochemical cycling and community composition dynamics. Among the many diverse viruses from terrestrial environments found in environmental waters, some are plant, animal, and/or human pathogens. The majority of pathogenic viral species found in waters are very stable and can survive outside host cells for long periods. The occurrence of such viruses in environmental waters has raised concerns because of the confirmation of the infectivity of waterborne viruses even at very low concentrations. This chapter focuses mainly on the survival of human, animal, and plant pathogenic viruses in aqueous environments, the possibility of their water-mediated transmission, the ecological implications of viruses in water, the methods adapted for detecting such viruses, and how to minimize the risk of viruses spreading through water.