Naturally Occurring Yeasts Associated with Thaumatotibia leucotreta Can Enhance the Efficacy of the Cryptophlebia Leucotreta Granulovirus.

Yeasts associated with lepidopteran pests have been shown to play a role in their survival, development, and oviposition preference. It has been demonstrated that combining these yeasts with existing biological control agents can enhance their efficacy. The tortricid Thaumatotibia leucotreta is a phytosanitary pest in the South African citrus industry, with the baculovirus Cryptophlebia leucotreta granulovirus (CrleGV) being one of the components that can control this pest. Several yeast species were shown to be associated with T. leucotreta larvae, which affected their behaviour and development. A series of detached fruit bioassays were performed to determine whether the combination of yeast with CrleGV enhances its efficacy. These assays included determining the optimal yeast/virus ratio, testing all isolated yeast species in combination with CrleGV, and further improving yeast/virus formulation by adding an adjuvant. The optimal yeast concentration to use alongside CrleGV was determined to be 106 cells·mL-1. Pichia kluyveri, P. kudriavzevii, Kluyveromyces marxianus, and Saccharomyces cerevisiae in combination with CrleGV reduced larval survival compared to CrleGV alone. The addition of molasses and BREAK-THRU® S 240 to P. kudriavzevii and S. cerevisiae in combination with CrleGV did not notably improve their effectiveness; however, there was an observed decrease in larval survival. In future studies, field trials will be conducted with combinations of CrleGV and P. kudriavzevii or S. cerevisiae to investigate whether these laboratory findings can be replicated in orchard conditions.

Variable Region Sequences Influence 16S rRNA Performance.

16S rRNA gene sequences are commonly analyzed for taxonomic and phylogenetic studies because they contain variable regions that can help distinguish different genera. However, intra-genus distinction using variable region homology is often impossible due to the high overall sequence identities among closely related species, even though some residues may be conserved within respective species. Using a computational method that included the allelic diversity within individual genomes, we discovered that certain Escherichia and Shigella species can be distinguished by a multi-allelic 16S rRNA variable region single nucleotide polymorphism (SNP). To evaluate the performance of 16S rRNAs with altered variable regions, we developed an in vivo system that measures the acceptance and distribution of variant 16S rRNAs into a large pool of natural versions supporting normal translation and growth. We found that 16S rRNAs containing evolutionarily disparate variable regions were underpopulated both in ribosomes and in active translation pools, even for an SNP. Overall, this study revealed that variable region sequences can substantially influence the performance of 16S rRNAs and that this biological constraint can be leveraged to justify refining taxonomic assignments of variable region sequence data. IMPORTANCE This study reevaluates the notion that 16S rRNA gene variable region sequences are uninformative for intra-genus classification and that single nucleotide variations within them have no consequence to strains that bear them. We demonstrated that the performance of 16S rRNAs in Escherichia coli can be negatively impacted by sequence changes in variable regions, even for single nucleotide changes that are native to closely related Escherichia and Shigella species; thus, biological performance is likely constraining the evolution of variable regions in bacteria. Further, the native nucleotide variations we tested occur in all strains of their respective species and across their multiple 16S rRNA gene copies, suggesting that these species evolved beyond what would be discerned from a consensus sequence comparison. Therefore, this work also reveals that the multiple 16S rRNA gene alleles found in most bacteria can provide more informative phylogenetic and taxonomic detail than a single reference allele.

Efficacy of Various Low Temperature and Exposure Time Combinations for Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae) Larvae.

A systems approach was developed as an alternative to a standalone quarantine disinfestation treatment for Thaumatotibia leucotreta in citrus fruit exported from South Africa. The systems approach consists of three measures: pre and postharvest controls and measurements, postpacking inspection, and postharvest exposure to low temperatures. Different cold treatment conditions with a range of efficacy levels can be used for this last measure. A series of trials reported here evaluated the efficacy of seven temperatures ranging from 0 to 5°C for durations from 14 d to 26 d. Mortality of the most cold-tolerant larval stages of T. leucotreta was determined. Temperatures of 0, 1, 2, and 3°C for 16, 19, 20, and 24 d respectively, induced 100% mortality of the tested populations. Probit 9 level treatment efficacy was achieved at 0 and 1°C for 16 and 19 d respectively. Mortalities higher than 90% were obtained with temperatures of 4, 4.5, and 5°C, after exposure for the longer durations. We demonstrated a significant difference in cold-induced insecticidal efficacy between 1, 2, 3, and 4°C. There was no significant difference in insecticidal efficacy between 4 and 4.5°C, but both of these temperatures were more efficacious than 5°C. The results of this study are valuable to support the use of cold treatment conditions with lower risk of fruit chilling injury in an effective systems approach, where the cold treatment efficacy can be augmented with other components of the systems approach.

E-Cigarette Aerosol Exposure Favors the Growth and Colonization of Oral Streptococcus mutans Compared to Commensal Streptococci.

E-cigarettes (e-cigs) have drastically increased in popularity during the last decade, especially among teenagers. While recent studies have started to explore the effect of e-cigs in the oral cavity, little is known about their effects on the oral microbiota and how they could affect oral health and potentially lead to disease, including periodontitis and head and neck cancers. To explore the impact of e-cigs on oral bacteria, we selected members of the genus Streptococcus, which are abundant in the oral cavity. We exposed the commensals Streptococcus sanguinis and Streptococcus gordonii and the opportunistic pathogen Streptococcus mutans, best known for causing dental caries, to e-liquids and e-cig aerosols with and without nicotine and with and without menthol flavoring and measured changes in growth patterns and biofilm formation. Our results demonstrate that e-cig aerosols hindered the growth of S. sanguinis and S. gordonii, while they did not affect the growth of S. mutans. We also show that e-cig aerosols significantly increased biofilm formation by S. mutans but did not affect the biofilm formation of the two commensals. We found that S. mutans exhibits higher hydrophobicity and coaggregation abilities along with higher attachment to OKF6 cells than S. sanguinis and S. gordonii. Therefore, our data suggest that e-cig aerosols have the potential to dysregulate oral bacterial homeostasis by suppressing the growth of commensals while enhancing the biofilm formation of the opportunistic pathogen S. mutans. This study highlights the importance of understanding the consequences of e-cig aerosol exposure on selected commensals and pathogenic species. Future studies modeling more complex communities will provide more insight into how e-cig aerosols and vaping affect the oral microbiota. IMPORTANCE Our study shows that e-cigarette aerosol exposure of selected bacteria known to be residents of the oral cavity hinders the growth of two streptococcal commensals while enhancing biofilm formation, hydrophobicity, and attachment for the pathogen S. mutans. These results indicate that e-cigarette vaping could open a niche for opportunistic bacteria such as S. mutans to colonize the oral cavity and affect oral health.

Mutualism between Gut-Borne Yeasts and Their Host, Thaumatotibia leucotreta, and Potential Usefulness in Pest Management

Thaumatotibia leucotreta is endemic to southern Africa and is highly significant for various fruit industries, including the South African citrus industry, due to its classification as a phytosanitary pest. Mutualistic associations between C. pomonella, closely related to T. leucotreta, and yeasts have previously been described and reported to reduce larval mortality and enhance larval development. Here, we determined which yeast species occur naturally in the gut of T. leucotreta larvae and investigated whether any of the isolated yeast species affect their behaviour and development. Navel oranges infested with T. leucotreta larvae were collected from geographically distinct provinces in South Africa, and the larvae were processed for analysis of naturally occurring associated yeasts. Six yeast species were isolated and identified from the guts of these T. leucotreta larvae via PCR amplification and sequencing of the ITS region of rDNA and D1/D2 domain of large ribosomal subunit. Larval development and attraction assays were conducted, and T. leucotreta larvae that fed on Navel oranges inoculated with yeast had accelerated developmental periods and reduced mortality rates. Neonate T. leucotreta were also attracted to YPD broth cultures inoculated with yeast for feeding. Oviposition preference assays were conducted with adult T. leucotreta females. Navel oranges inoculated with yeast were shown to influence the oviposition preference of adult females. Yeasts harbour the potential for use in biocontrol, especially when combined with other well-established control methods. This study provides a platform for future research into incorporating yeast with current biological control agents as a novel option for controlling T. leucotreta in the field.

E-Cigarette Aerosols Promote Oral S. aureus Colonization by Delaying an Immune Response and Bacterial Clearing.

E-cigarette (e-cig) vapor has been shown to play a pathological role in oral health and alter the oral microbiota, providing growth advantages for opportunistic pathogens. Enrichment of Staphylococcus aureus, a commensal resident in the oral cavity, correlates with the progression of periodontal disease, suggesting a role as an opportunistic pathogen. Environmental conditions, such as cigarette smoke, are known to increase S. aureus virulence, yet the role of S. aureus in periodontitis and oral preneoplasia is unknown. We exposed oral epithelial cells to e-cig aerosols and showed a dose-dependent cell viability reduction, regardless of nicotine content, in a possible attempt to repair DNA damage, as measured by pH2AX. S. aureus attachment to oral epithelial cells and bacterial biofilm formation were enhanced upon e-cig exposure, indicating an increased capacity for oral colonization. Mechanistically, e-cig aerosol exposure resulted in an immunosuppression, as determined by a reduction in IL8, IL6, and IL1ß secretion by oral epithelial cells during co-culture with S. aureus. Consistent with this, e-cig vape reduced the oral epithelial cell clearance of S. aureus. Furthermore, we observed an increased expression of the inflammatory regulator COX2. This work suggests that e-cigs promote S. aureus colonization and modulate the oral inflammatory response, possibly promoting oral periodontitis and preneoplasia.

Microbial Communities in Retail Draft Beers and the Biofilms They Produce.

In the beer brewing industry, microbial spoilage presents a consistent threat that must be monitored and controlled to ensure the palatability of a finished product. Many of the predominant beer spoilage microbes have been identified and characterized, but the mechanisms of contamination and persistence remain an open area of study. Postproduction, many beers are distributed as kegs that are attached to draft delivery systems in retail settings where ample opportunities for microbial spoilage are present. As such, restaurants and bars can experience substantial costs and downtime for cleaning when beer draft lines become heavily contaminated. Spoilage monitoring on the retail side of the beer industry is often overlooked, yet this arena may represent one of the largest threats to the profitability of a beer if its flavor profile becomes substantially distorted by contaminating microbes. In this study, we sampled and cultured microbial communities found in beers dispensed from a retail draft system to identify the contaminating bacteria and yeasts. We also evaluated their capability to establish new biofilms in a controlled setting. Among four tested beer types, we identified over a hundred different contaminant bacteria and nearly 20 wild yeasts. The culturing experiments demonstrated that most of these microbes were viable and capable of joining new biofilm communities. These data provide an important reference for monitoring specific beer spoilage microbes in draft systems and we provide suggestions for cleaning protocol improvements. IMPORTANCE Beer production, packaging, and service are each vulnerable to contamination by microbes that metabolize beer chemicals and impart undesirable flavors, which can result in the disposal of entire batches. Therefore, great effort is taken by brewmasters to reduce and monitor contamination during production and packaging. A commonly overlooked quality control stage of a beer supply chain is at the retail service end, where beer kegs supply draft lines in bars and restaurants under nonsterile conditions. We found that retail draft line contamination is rampant and that routine line cleaning methods are insufficient to efficiently suppress beer spoilage. Thus, many customers unknowingly consume spoiled versions of the beers they consume. This study identified the bacteria and yeast that were resident in retail draft beer samples and also investigated their abilities to colonize tubing material as members of biofilm communities.

Crowdsourcing biocuration: The Community Assessment of Community Annotation with Ontologies (CACAO).

Experimental data about gene functions curated from the primary literature have enormous value for research scientists in understanding biology. Using the Gene Ontology (GO), manual curation by experts has provided an important resource for studying gene function, especially within model organisms. Unprecedented expansion of the scientific literature and validation of the predicted proteins have increased both data value and the challenges of keeping pace. Capturing literature-based functional annotations is limited by the ability of biocurators to handle the massive and rapidly growing scientific literature. Within the community-oriented wiki framework for GO annotation called the Gene Ontology Normal Usage Tracking System (GONUTS), we describe an approach to expand biocuration through crowdsourcing with undergraduates. This multiplies the number of high-quality annotations in international databases, enriches our coverage of the literature on normal gene function, and pushes the field in new directions. From an intercollegiate competition judged by experienced biocurators, Community Assessment of Community Annotation with Ontologies (CACAO), we have contributed nearly 5,000 literature-based annotations. Many of those annotations are to organisms not currently well-represented within GO. Over a 10-year history, our community contributors have spurred changes to the ontology not traditionally covered by professional biocurators. The CACAO principle of relying on community members to participate in and shape the future of biocuration in GO is a powerful and scalable model used to promote the scientific enterprise. It also provides undergraduate students with a unique and enriching introduction to critical reading of primary literature and acquisition of marketable skills.

Exopolysaccharide anchoring creates an extreme resistance to sedimentation.

By evolving strains of E. coli that hyper-resist sedimentation, we discovered an uncharacterized mechanism that bacteria can use to remain in suspension indefinitely without expending energy. This unusual phenotype was traced to the anchoring of long colanic acid polymers (CAP) that project from the cell surface. Although each characterized mutant activated this same mechanism, the genes responsible and the strengths of the phenotypes varied. Mutations in rcsC, lpp, igaA, or the yjbEFGH operon were sufficient to stimulate sedimentation resistance, while mutations altering the cps promoter, cdgI, or yjbF provided phenotypic enhancements. The sedimentation resistances changed in response to temperature, growth phase, and carbon source and each mutant exhibited significantly reduced biofilm formation. We discovered that the degree of colony mucoidy exhibited by these mutants was not related to the degree of Rcs pathways activation or to the amount of CAP that was produced; rather, it was related to the fraction of CAP that was shed as a true exopolysaccharide. Therefore, these and other mutations that activate this phenotype are likely to be absent from genetic screens that relied on centrifugation to harvest bacteria. We also found that this anchored CAP form is not linked to LPS cores and may not be attached to the outer membrane.IMPORTANCEBacteria can partition in aqueous environments between surface-dwelling, planktonic, sedimentary, and biofilm forms. Residence in each location provides an advantage depending on nutritional and environmental stresses and a community of a single species is often observed to be distributed throughout two or more of these niches. Another adaptive strategy is to produce an extracellular capsule, which provides an environmental shield for the microbe and can allow escape from predators and immune systems. We discovered that bacteria can either shed or stably anchor capsules to dramatically alter their propensity to sediment. The degree to which the bacteria anchor their capsule is controlled by a stress sensing system, suggesting that anchoring may be used as an adaptive response to severe environmental challenges.

Modeling Small Structural and Environmental Differences in Solids with 15 N†NMR Chemical Shift Tensors.

The ability to theoretically predict accurate NMR chemical shifts in solids is increasingly important due to the role such shifts play in selecting among proposed model structures. Herein, two theoretical methods are evaluated for their ability to assign 15 N shifts from guanosine dihydrate to one of the two independent molecules present in the lattice. The NMR data consist of 15 N shift tensors from 10 resonances. Analysis using periodic boundary or fragment methods consider a benchmark dataset to estimate errors and predict uncertainties of 5.6 and 6.2 ppm, respectively. Despite this high accuracy, only one of the five sites were confidently assigned to a specific molecule of the asymmetric unit. This limitation is not due to negligible differences in experimental data, as most sites exhibit differences of >6.0 ppm between pairs of resonances representing a given position. Instead, the theoretical methods are insufficiently accurate to make assignments at most positions.

Biological Control of a Phytosanitary Pest (Thaumatotibia leucotreta): A Case Study.

Thaumatotibia leucotreta, known as the false codling moth, is a pest of citrus and other crops in sub-Saharan Africa. As it is endemic to this region and as South Africa exports most of its citrus around the world, T. leucotreta has phytosanitary status for most markets. This means that there is zero tolerance for any infestation with live larvae in the market. Consequently, control measures prior to exporting must be exemplary. Certain markets require a standalone postharvest disinfestation treatment for T. leucotreta. However, the European Union accepts a systems approach, consisting of three measures and numerous components within these measures. Although effective preharvest control measures are important under all circumstances, they are most critical where a standalone postharvest disinfestation treatment is not applied, such as within a systems approach. Conventional wisdom may lead a belief that effective chemical control tools are imperative to achieve this end. However, we demonstrate that it is possible to effectively control T. leucotreta to a level acceptable for a phytosanitary market, using only biological control tools. This includes parasitoids, predators, microbial control, semiochemicals, and sterile insects. Simultaneously, on-farm and environmental safety is improved and compliance with the increasing stringency of chemical residue requirements imposed by markets is achieved.

Ribosome collisions alter frameshifting at translational reprogramming motifs in bacterial mRNAs.

Translational frameshifting involves the repositioning of ribosomes on their messages into decoding frames that differ from those dictated during initiation. Some messenger RNAs (mRNAs) contain motifs that promote deliberate frameshifting to regulate production of the encoded proteins. The mechanisms of frameshifting have been investigated in many systems, and the resulting models generally involve single ribosomes responding to stimulator sequences in their engaged mRNAs. We discovered that the abundance of ribosomes on messages containing the IS3, dnaX, and prfB frameshift motifs significantly influences the levels of frameshifting. We show that this phenomenon results from ribosome collisions that occur during translational stalling, which can alter frameshifting in both the stalled and trailing ribosomes. Bacteria missing ribosomal protein bL9 are known to exhibit a reduction in reading frame maintenance and to have a strong dependence on elongation factor P (EFP). We discovered that ribosomes lacking bL9 become compacted closer together during collisions and that the E-sites of the stalled ribosomes appear to become blocked, which suggests subsequent transpeptidation in transiently stalled ribosomes may become compromised in the absence of bL9. In addition, we determined that bL9 can suppress frameshifting of its host ribosome, likely by regulating E-site dynamics. These findings provide mechanistic insight into the behavior of colliding ribosomes during translation and suggest naturally occurring frameshift elements may be regulated by the abundance of ribosomes relative to an mRNA pool.

Genome Analysis of A Novel South African Cydia pomonella granulovirus (CpGV-SA) with Resistance-Breaking Potential.

The complete genome of an endemic South African Cydia pomonella granulovirus isolate was sequenced and analyzed. Several missing or truncated open reading frames (ORFs) were identified, including a 24 bp deletion in the pe38 gene which is reported to be associated with type I resistance-breaking potential. Comparison of single nucleotide polymorphisms (SNPs) with five other fully sequenced CpGV isolates identified 67 unique events, 47 of which occurred within ORFs, leading to several amino acid changes. Further analysis of single nucleotide variations (SNVs) within CpGV-SA revealed that this isolate consists of mixed genotypes. Phylogenetic analysis using complete genome sequences placed CpGV-SA basal to M, I12 and E2 and distal to S and I07 but with no distinct classification into any of the previously defined CpGV genogroups. These results suggest that CpGV-SA is a novel and genetically distinct isolate with significant potential as a biopesticide for management of codling moth (CM), not only in South Africa, but potentially in other pome fruit producing countries, particularly where CM resistance to CpGV has been reported.

Cryptophlebia peltastica Nucleopolyhedrovirus Is Highly Infectious to Codling Moth Larvae and Cells.

Cydia pomonella granulovirus (CpGV) is a cornerstone of codling moth (Cydia pomonella) control in integrated and organic pome fruit production, though different types of resistance to CpGV products have been recorded in codling moth field populations in Europe for several years. Recently, a novel baculovirus named Cryptophlebia peltastica nucleopolyhedrovirus (CrpeNPV) was isolated from a laboratory culture of the litchi moth, Cryptophlebia peltastica, in South Africa. Along with CpGV, it is the third known baculovirus that is infectious to codling moth. In the present study, parameters of infectiveness of CrpeNPV, such as the median lethal concentration and median survival time, were determined for codling moth larvae susceptible or resistant to CpGV. In addition, the permissiveness of a codling moth cell line with respect to infection by CrpeNPV budded virus was demonstrated by infection and gene expression studies designed to investigate the complete replication cycle. Investigations of the high degree of virulence of CrpeNPV for codling moth larvae and cells are of high significant scientific and economic value and may offer new strategies for the biological control of susceptible and resistant populations of codling moth.IMPORTANCE The emergence of codling moth populations resistant to commercially applied isolates of CpGV is posing an imminent threat to organic pome fruit production. Very few CpGV isolates are left that are able to overcome the reported types of resistance, emphasizing the demand for new and highly virulent baculoviruses. Here we report the recently discovered CrpeNPV as highly infectious to all types of resistant codling moth populations with a high speed of killing, making it a promising candidate baculovirus in fighting the spread of resistant codling moth populations.

New Method for Differentiation of Granuloviruses (Betabaculoviruses) Based on Real-Time Polymerase Chain Reaction (Real-Time PCR).

Baculoviridae is a highly diverse family of rod-shaped viruses with double-stranded DNA. To date, almost 100 species have had their complete genomic sequences deposited in the GenBank database, a quarter of which comprises granuloviruses (GVs). Many of the genomes are sequenced using next-generation sequencing, which is currently considered the best method for characterizing new species, but it is time-consuming and expensive. Baculoviruses form a safe alternative to overused chemical pesticides and therefore there is a constant need for identifying new species that can be active components of novel biological insecticides. In this study, we have described a fast and reliable method for the detection of new and differentiation of previously analyzed granulovirus species based on a real-time polymerase chain reaction (PCR) technique with melting point curve analysis. The sequences of highly conserved baculovirus genes, such as granulin and late expression factors 8 and 9 (lef-8 and lef-9), derived from GVs available to date have been analyzed and used for degenerate primer design. The developed method was tested on a representative group of eight betabaculoviruses with comparisons of melting temperatures to allow for quick and preliminary granulovirus detection. The proposed real-time PCR procedure may be a very useful tool as an easily accessible screening method in a majority of laboratories.

Microbial control of phytophagous invertebrate pests in South Africa: Current status and future prospects.

Invertebrate pests pose a significant threat to food security on the African continent. In response, South Africa has become one of the largest importers of chemical pesticides in sub-Saharan Africa, with several hundred active ingredients registered. To address the over-reliance on such chemicals, the South African Department of Agriculture, Forestry and Fisheries (DAFF) has eliminated or restricted several pesticides since the late 1970s. The recent launch of the South African National Bio-Economy Strategy and establishment of the South African Bioproducts Organisation (SABO), together with new guidelines for registration of biopesticides in 2015, also support this endeavour. Concurrently, entomopathogen-related research and bioproduct development has increased over the past decade. Currently, 31 products (seven manufactured locally) are registered under the Fertilizers, Farm Feeds, Agricultural Remedies and Stock Remedies Act 36 of 1947. Commercially important microbes include Beauveria bassiana (Cordycipitaceae), Metarhizium anisopliae (Clavicipitaceae), Cydia pomonella granulovirus, Cryptophlebia leucotreta granulovirus, Helicoverpa armigera nucleopolyhedrovirus (Baculoviridae) and Bacillus thuringiensis subsp. kurstaki and B. thuringiensis subsp. aizawai (Bacillaceae). Both parasitic and entomopathogenic nematodes (EPNs) show potential for development as bioinsecticides with one commercial EPN product, based on Heterorhabditis bacteriophora (Heterorhabditidae), registered under the Act. Rapid scientific progression, supported by a favourable legislative environment, should facilitate further advances in microbial control of phytophagous invertebrate pests in South Africa.

Development of a Postharvest Cold Treatment for Cryptophlebia peltastica (Lepidoptera: Tortricidae) for Export of Litchis From South Africa.

The litchi moth, Cryptophlebia peltastica (Meyrick) (Lepidoptera: Tortricidae), is endemic to sub-Saharan Africa and certain Indian Ocean islands. It is an important pest of litchis and to a lesser extent macadamias. Litchis are exported to certain markets that consider C. peltastica as a phytosanitary pest. Consequently, an effective postharvest phytosanitary treatment is required. This study sought to develop a cold disinfestation treatment for this purpose. First, it was established that the fifth instar was the most cold-tolerant larval stage, as it was the only instar for which there was still some survival after 12 d at 1°C. It was then determined that cold treatment trials could be conducted in artificial diet, as there was no survival of fifth instar C. peltastica in litchis after only 9 d at 1°C, whereas it took 15 d at this temperature before no survival of fifth instar C. peltastica was recorded in artificial diet. Consequently, cold susceptibility of fifth instar C. peltastica and the most cold-tolerant larval stages (fourth and fifth instar) of false codling moth, Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae), were compared in artificial diet. There was no survival of C. peltastica after 13 d at 1°C, whereas this was only so for T. leucotreta after 16 d. Consequently, it can be concluded that any cold treatment that has been proven effective against T. leucotreta would be as effective against C. peltastica. Finally, it was confirmed that the cold susceptibility of T. leucotreta in artificial diet did not overestimate the effect of cold on T. leucotreta larvae in litchis.

A Solvent-Free Approach for Converting Cellulose Waste into Volatile Organic Compounds with Endophytic Fungi.

Simple sugars produced from a solvent-free mechanocatalytic degradation of cellulose were evaluated for suitability as a growth medium carbon source for fungi that produce volatile organic compounds. An endophytic Hypoxylon sp. (CI-4) known to produce volatiles having potential value as fuels was initially evaluated. The growth was obtained on a medium containing the degraded cellulose as the sole carbon source, and the volatile compounds produced were largely the same as those produced from a conventional dextrose/starch diet. A second Hypoxylon sp. (BS15) was also characterized and shown to be phylogenetically divergent from any other named species. The degraded cellulose medium supported the growth of BS15, and approximately the same quantity of the volatile compounds was produced as from conventional diets. Although the major products from BS15 grown on the degraded cellulose were identical to those from dextrose, the minor products differed. Neither CI-4 or BS15 exhibited growth on cellulose that had not been degraded. The extraction of volatiles from the growth media was achieved using solid-phase extraction in order to reduce the solvent waste and more efficiently retain compounds having low vapor pressures. A comparison to more conventional liquid⁻liquid extraction demonstrated that, for CI-4, both methods gave similar results. The solid-phase extraction of BS15 retained a significantly larger variety of the volatile compounds than did the liquid⁻liquid extraction. These advances position the coupling of solvent-free cellulose conversion and endophyte metabolism as a viable strategy for the production of important hydrocarbons.

Morphological, genetic and biological characterisation of a novel alphabaculovirus isolated from Cryptophlebia peltastica (Lepidoptera: Tortricidae).

Cryptophlebia peltastica is an agricultural pest of litchis and macadamias in South Africa with phytosanitary status for certain markets. Current control methods rely on chemical, cultural and classical biological control. However, a microbial control option has not been developed. An Alphabaculovirus from C. peltastica was recovered from a laboratory reared colony and morphologically characterised by transmission electron microscopy (TEM). Analysis of occlusion bodies indicated a single NPV (SNPV) varying in size from 421 to 1263 nm. PCR amplification and sequencing of the polh gene region using universal primers followed by BLAST analysis revealed a 93% similarity to a partial polh gene sequence from Epinotia granitalis NPV. Further genetic characterisation involving single restriction endonuclease (REN) digestion of genomic DNA was carried out to generate profiles for comparison against other baculovirus species and potential new isolates of the same virus. The complete genome of the virus was sequenced, assembled and analysed for a more comprehensive genetic analysis. The genome was 115728 base pairs (bp) in length with a GC content of 37.2%. A total of 126 open reading frames (ORFs) were identified with minimal overlap and no preference in orientation. Bioassays were used to determine the virulence of the NPV against C. peltastica. The NPV was virulent against C. peltastica with an LC50 value of 6.46 × 103 OBs/ml and an LC90 value of 2.46 × 105 OBs/ml, and time mortality ranging between 76.32 h and 93.49 h. This is the first study to describe the isolation and genetic characterisation of a novel SNPV from C. peltastica, which has potential for development into a biopesticide for the control of this pest in South Africa.

New Method for Differentiation of Granuloviruses (Betabaculoviruses) Based on Multitemperature Single Stranded Conformational Polymorphism.

Baculoviruses have been used as biopesticides for decades. Recently, due to the excessive use of chemical pesticides there is a need for finding new agents that may be useful in biological protection. Sometimes few isolates or species are discovered in one host. In the past few years, many new baculovirus species have been isolated from environmental samples, thoroughly characterized and thanks to next generation sequencing methods their genomes are being deposited in the GenBank database. Next generation sequencing (NGS) methodology is the most certain way of detection, but it has many disadvantages. During our studies, we have developed a method based on Polymerase chain reaction (PCR) followed by Multitemperature Single Stranded Conformational Polymorphism (MSSCP) which allows for distinguishing new granulovirus isolates in only a few hours and at low-cost. On the basis of phylogenetic analysis of betabaculoviruses, representative species have been chosen. The alignment of highly conserved genes-granulin and late expression factor-9, was performed and the degenerate primers were designed to amplify the most variable, short DNA fragments flanked with the most conserved sequences. Afterwards, products of PCR reaction were analysed by MSSCP technique. In our opinion, the proposed method may be used for screening of new isolates derived from environmental samples.