Non-pathogenic Isolates Research Articles

Resource capture and competitive ability of non-pathogenic Pseudogymnoascus spp. and P. destructans, the cause of white-nose syndrome in bats

White-nose syndrome (WNS) is a devastating fungal disease that has been causing the mass mortality of hibernating bats in North America since 2006 and is caused by the psychrophilic dermatophyte Pseudogymnoascus destructans. Infected bats shed conidia into hibernaculum sediments and surfaces, but it is unknown if P. destructans can form stable, reproductive populations outside its bat hosts. Previous studies have found non-pathogenic Pseudogymnoascus in bat hibernacula, and these fungi may provide insight into the natural history of P. destructans. We compared the relatedness, resource capture, and competitive ability of non-pathogenic Pseudogymnoascus isolates with P. destructans to determine if they have similar adaptations for survival in hibernacula sediment. All non-pathogenic Pseudogymnoascus isolates grew faster, utilized a broader range of substrates with higher efficiency, and were generally more resistant to antifungals compared to P. destructans. All isolates also showed the ability to displace P. destructans in co-culture assays, but only some produced extractible antifungal metabolites. These results suggest that P. destructans would perform poorly in the same environmental niche as non-pathogenic Pseudogymnoascus, and must have an alternative saprophytic survival strategy if it establishes active populations in hibernaculum sediment and non-host surfaces.

Effector Gene Suites in Some Soil Isolates of Fusarium oxysporum Are Not Sufficient Predictors of Vascular Wilt in Tomato.

Seventy-four Fusarium oxysporum soil isolates were assayed for known effector genes present in an F. oxysporum f. sp. lycopersici race 3 tomato wilt strain (FOL MN-25) obtained from the same fields in Manatee County, Florida. Based on the presence or absence of these genes, four haplotypes were defined, two of which represented 96% of the surveyed isolates. These two most common effector haplotypes contained either all or none of the assayed race 3 effector genes. We hypothesized that soil isolates with all surveyed effector genes, similar to FOL MN-25, would be pathogenic toward tomato, whereas isolates lacking all effectors would be nonpathogenic. However, inoculation experiments revealed that presence of the effector genes alone was not sufficient to ensure pathogenicity on tomato. Interestingly, a nonpathogenic isolate containing the full suite of unmutated effector genes (FOS 4-4) appears to have undergone a chromosomal rearrangement yet remains vegetatively compatible with FOL MN-25. These observations confirm the highly dynamic nature of the F. oxysporum genome and support the conclusion that pathogenesis among free-living populations of F. oxysporum is a complex process. Therefore, the presence of effector genes alone may not be an accurate predictor of pathogenicity among soil isolates of F. oxysporum.

Yersinia spp. in Wild Rodents and Shrews in Finland.

Yersinia enterocolitica and Yersinia pseudotuberculosis are important zoonotic bacteria causing human enteric yersiniosis commonly reported in Europe. All Y. pseudotuberculosis strains are considered pathogenic, while Y. enterocolitica include both pathogenic and nonpathogenic strains which can be divided into six biotypes (1A, 1B, and 2-5) and about 30 serotypes. The most common types causing yersiniosis in Europe are Y. enterocolitica bioserotypes 4/O:3 and 2/O:9. Strains belonging to biotype 1A are considered as nonpathogenic because they are missing important virulence genes like the attachment-invasion-locus (ail) gene in the chromosome and the virulence plasmid. The role of wild small mammals as a reservoir of enteropathogenic Yersinia spp. is still obscure. In this study, the presence of Yersinia spp. was examined from 1840 wild small mammals, including voles, mice, and shrews, trapped in Finland during a 7-year period. We isolated seven Yersinia species. Y. enterocolitica was the most common species, isolated from 8% of the animals; while most of these isolates represented nonpathogenic biotype 1A, human pathogenic bioserotype 2/O:9 was also isolated from a field vole. Y. pseudotuberculosis of bioserotype 1/O:2 was isolated from two shrews. The ail gene, which is typically only found in the isolates of biotypes 1B and 2-5 associated with yersiniosis, was frequently (23%) detected in the nonpathogenic isolates of biotype 1A and sporadically (6%) in Yersinia kristensenii isolates. Our results suggest that wild small mammals, especially voles, may serve as carriers for ail-positive Y. enterocolitica 1A and Y. kristensenii. We also demonstrate that voles and shrews sporadically excrete pYV-positive Y. enterocolitica 2/O:9 and Y. pseudotuberculosis 1/O:2, respectively, in their feces and, thus, can serve as a contamination source for vegetables by contaminating the soil.

Variability among isolates of Fusarium udum and the effect on progression of wilt in pigeonpea

Vascular wilt (caused by Fusarium udum) is the most important disease of pigeonpea. Breeding strategies are needed to develop durable resistance against this pathogen. Knowledge of the genetic and pathogenic variability among isolates of F. udum is essential for effective deployment of resistance. Thirteen isolates of F. udum, collected from three regions of India were studied using cultural characteristics, molecular variability and pathogenicity. The isolates of F. udum showed variability in aerial and radial mycelium growth, colour of mycelia, substrate pigmentation, length and septation of macro- and microconidia. Amplified fragment length polymorphism (AFLP) analysis of the isolates determined seven phylogenetic groups. Pathogenesis and progress of wilt on susceptible pigeonpea cultivars varied and pathogenic isolates from the same phylogenetic group exhibited similarities in timing of infection and colonization. Non-pathogenic isolates formed separate clusters in the phylogenetic tree. The timing of fungal invasion, vascular clogging, drooping of shoots, and wilt establishment were demonstrated for the first time in pigeonpea. Vascular clogging occurred with susceptible cultivars at 78–84, 54–60, 48–60 and 36–48 h post inoculation (HPI) when inoculated with isolates of F. udum from AFLP groups 1, 3, 6 and 7, respectively. Subsequently, susceptible cultivars inoculated with isolates from the same groups exhibited wilt at 144–168, 120–144, 96–120 and 72–96 HPI, respectively. This study contributes to characterising vascular wilt of pigeonpea.

The Population of Fusarium oxysporum f. sp. fragariae, Cause of Fusarium Wilt of Strawberry, in California.

The objectives of this study were to investigate the structure of the population of Fusarium oxysporum f. sp. fragariae in California and to evaluate methods for its detection. Fifty-nine isolates of F. oxysporum f. sp. fragariae were obtained from diseased strawberry plants and their identity was confirmed by pathogenicity testing. The full nuclear ribosomal intergenic spacer (IGS) and elongation factor 1-α gene (EF-1α) were amplified by polymerase chain reaction (PCR) and sequenced to elucidate phylogenetic relationships among isolates. IGS and EF-1α sequences revealed three main lineages, which corresponded to three somatic compatibility groups. Primers designed to detect F. oxysporum f. sp. fragariae in Japan amplified a 239-bp product from 55 of 59 California isolates of F. oxysporum f. sp. fragariae and from no nonpathogenic isolates of F. oxysporum. The sequence of this PCR product was identical to the sequence obtained from F. oxysporum f. sp. fragariae isolates in Japan. Intensive sampling at two locations in California showed results of tests based on PCR and somatic compatibility to be in agreement for 97% (257 of 264) of isolates tested. Our findings revealed considerable diversity in the California population of F. oxysporum f. sp. fragariae, and indications that horizontal gene transfer may have occurred.

The Ever Changing Face of Antibiotic Resistance: Prevailing Problems and Preventive Measures.

Antibiotic resistance is a global problem that presents significant risk to human health. Driven by selective pressure of antimicrobial agents, spontaneous mutation, recombination and horizontal gene transfer events, inappropriate antibiotic prescribing and use outside healthcare settings has increased their impact on healthcare system. Increasing risk for human health lead us to study resistance development mechanisms, associated factors that increase dissemination of resistance genes along with information of imperative measures necessary to curtail the growing menace. In this article, we emphasized on the state of knowledge regarding imprudent use of antibiotics that act as promoters of resistance development. For this, literature based search for articles and entries related to antimicrobial resistance was done. With ample of data available, selected was performed for the epidemiological and clinical based study to curtail the facts present in these data sets so as to get accurate and important information. Resistance mediated by different determinants such as TEM, SHV, OXA and CTX-M, methods of mobilization that increase spread across species and as such failure to available treatment regimens was studied. Addition to detection methods, information of the inhibitors and natural substance useful in mitigating the effect of multidrug resistance was included to strategies the policies and plans for restricting their spread. As intervention to this growing problem, modified use of antimicrobial agents, employment of different formulations of herbs along with public health interventions in restricting antibiotic use, are believed to be of great help in restricting their dissemination and as such spread to non-pathogenic bacterial isolates.

Administration of non-pathogenic isolates of Escherichia coli and Clostridium perfringens type A to piglets in a herd affected with a high incidence of neonatal diarrhoea

A bacterial cocktail of living strains of Clostridium perfringens type A (CPA) without β2-toxin gene and non-pathogenic Escherichia coli was administered orally to newborn piglets before first colostrum intake and on 2 consecutive days on a farm with a high incidence of diarrhoea and antibiotic treatment in suckling piglets associated with E. coli and CPA. This clinical field study was driven by the hypothetic principle of competitive exclusion of pathogenic bacteria due to prior colonization of the gut mucosal surface by non-pathogenic strains of the same bacterial species with the aim of preventing disease. Although CPA strains used in this study did not produce toxins in vitro, their lack of pathogenicity cannot be conclusively confirmed. The health status of the herd was impaired by a high incidence of postpartum dysgalactia syndrome in sows (70%) and a high incidence of neonatal diarrhoea caused by enterotoxigenic E. coli and CPA during the study. No obvious adverse effect of the bacterial treatment occurred. On average, more piglets were weaned in litters treated (P=0.009). Visual pathological alterations in the small intestinal wall were more frequent in dead piglets of the control group (P=0.004) and necrotizing enteritis was only found in that group. A higher average daily weight gain of piglets in the control group (P<0.001) may be due to an increased milk uptake due to less competition in the smaller litters. The bacterial cocktail was tested under field conditions for its potential to stabilize gut health status in suckling piglets before disease development due to colibacillosis and clostridial infections; however, the gut flora stabilizing effect of the bacterial cocktail was not clearly discernible in this study. Further basic research is needed to confirm the positive effects of the bacterial treatment used and to identify additional potential bacterial candidates for competitive exclusion.

Comparative Genomic Analysis of Two Serotype 1/2b Listeria monocytogenes Isolates from Analogous Environmental Niches Demonstrates the Influence of Hypervariable Hotspots in Defining Pathogenesis.

The vast majority of clinical human listeriosis cases are caused by serotype 1/2a, 1/2b, 1/2c, and 4b isolates of Listeria monocytogenes. The ability of L. monocytogenes to establish a systemic listeriosis infection within a host organism relies on a combination of genes that are involved in cell recognition, internalization, evasion of host defenses, and in vitro survival and growth. Recently, whole genome sequencing and comparative genomic analysis have proven to be powerful tools for the identification of these virulence-associated genes in L. monocytogenes. In this study, two serotype 1/2b strains of L. monocytogenes with analogous isolation sources, but differing infection abilities, were subjected to comparative genomic analysis. The results from this comparison highlight the importance of accessory genes (genes that are not part of the conserved core genome) in L. monocytogenes pathogenesis. In addition, a number of factors, which may account for the perceived inability of one of the strains to establish a systemic infection within its host, have been identified. These factors include the notable absence of the Listeria pathogenicity island 3 and the stress survival islet, of which the latter has been demonstrated to enhance the survival ability of L. monocytogenes during its passage through the host intestinal tract, leading to a higher infection rate. The findings from this research demonstrate the influence of hypervariable hotspots in defining the physiological characteristics of a L. monocytogenes strain and indicate that the emergence of a non-pathogenic isolate of L. monocytogenes may result from a cumulative loss of functionality rather than by a single isolated genetic event.

Resilient microorganisms in dust samples of the International Space Station-survival of the adaptation specialists.

BackgroundThe International Space Station (ISS) represents a unique biotope for the human crew but also for introduced microorganisms. Microbes experience selective pressures such as microgravity, desiccation, poor nutrient-availability due to cleaning, and an increased radiation level. We hypothesized that the microbial community inside the ISS is modified by adapting to these stresses.For this reason, we analyzed 8–12 years old dust samples from Russian ISS modules with major focus on the long-time surviving portion of the microbial community. We consequently assessed the cultivable microbiota of these samples in order to analyze their extremotolerant potential against desiccation, heat-shock, and clinically relevant antibiotics. In addition, we studied the bacterial and archaeal communities from the stored Russian dust samples via molecular methods (next-generation sequencing, NGS) and compared our new data with previously derived information from the US American ISS dust microbiome.ResultsWe cultivated and identified in total 85 bacterial, non-pathogenic isolates (17 different species) and 1 fungal isolate from the 8–12 year old dust samples collected in the Russian segment of the ISS. Most of these isolates exhibited robust resistance against heat-shock and clinically relevant antibiotics. Microbial 16S rRNA gene and archaeal 16S rRNA gene targeting Next Generation Sequencing showed signatures of human-associated microorganisms (Corynebacterium, Staphylococcus, Coprococcus etc.), but also specifically adapted extremotolerant microorganisms. Besides bacteria, the detection of archaeal signatures in higher abundance was striking.ConclusionsOur findings reveal (i) the occurrence of living, hardy microorganisms in archived Russian ISS dust samples, (ii) a profound resistance capacity of ISS microorganisms against environmental stresses, and (iii) the presence of archaeal signatures on board. In addition, we found indications that the microbial community in the Russian segment dust samples was different to recently reported US American ISS microbiota.Electronic supplementary materialThe online version of this article (doi:10.1186/s40168-016-0217-7) contains supplementary material, which is available to authorized users.

Longitudinal Comparison of Antibiotic Resistance in Diarrheagenic and Non-pathogenic Escherichia coli from Young Tanzanian Children.

Enteroaggregative, enteropathogenic, and enterotoxigenic Escherichia coli contribute significantly to the burden of diarrheal infections particularly in developing countries. Antibiotic resistance is increasingly common among bacterial pathogens including pathogenic E. coli. We assessed the relationship between pathogenic E. coli carriage and resistance to six antibiotics in E. coli isolated from young children in rural Tanzania. We surveyed temporal stability in antibiotic resistance in 2492 E. coli isolated from fecal samples obtained from young children in rural Tanzania collected over a 6 months period. Approximately half of the 377 children sampled were exposed to an azithromycin mass treatment program for trachoma control and half resided in control villages. Children were sampled at baseline, 1-, 3-, and 6 months following azithromycin treatment. We compared resistance to six antibiotics in pathogenic and non-pathogenic strains at the population level, within fecal specimens, and within individuals over time using chi-square tests, paired odds ratios, and logistic regression, respectively. Resistance to ampicillin and trimethoprim/sulfamethoxazole was highly prevalent (>65%). Resistance to 5 of 6 antibiotics tested and multi-drug resistance occurred more frequently in pathogenic isolates (p ≤ 0.001) within fecal specimens and overall. Azithromycin mass treatment exposure was significantly associated with increased odds of carriage of isolates resistant to erythromycin (OR 3.64, p < 0.001) and trimethoprim/sulfamethoxazole (OR 1.60, p < 0.05). Pathogenic isolates were approximately twice as likely to be resistant to erythromycin, ampicillin, or trimethoprim/sulfamethoxazole compared to non-pathogenic isolates from the same fecal specimen. The potential linkage between resistance and virulence in E. coli suggests hygiene and sanitation interventions aimed at reducing disease burden could play a role in controlling transmission of antibiotic resistance.

Overexpression of Differentially Expressed Genes Identified in Non-pathogenic and Pathogenic Entamoeba histolytica Clones Allow Identification of New Pathogenicity Factors Involved in Amoebic Liver Abscess Formation.

We here compared pathogenic (p) and non-pathogenic (np) isolates of Entamoeba histolytica to identify molecules involved in the ability of this parasite to induce amoebic liver abscess (ALA)-like lesions in two rodent models for the disease. We performed a comprehensive analysis of 12 clones (A1–A12) derived from a non-pathogenic isolate HM-1:IMSS-A and 12 clones (B1–B12) derived from a pathogenic isolate HM-1:IMSS-B. “Non-pathogenicity” included the induction of small and quickly resolved lesions while “pathogenicity” comprised larger abscess development that overstayed day 7 post infection. All A-clones were designated as non-pathogenic, whereas 4 out of 12 B-clones lost their ability to induce ALAs in gerbils. No correlation between ALA formation and cysteine peptidase (CP) activity, haemolytic activity, erythrophagocytosis, motility or cytopathic activity was found. To identify the molecular framework underlying different pathogenic phenotypes, three clones were selected for in-depth transcriptome analyses. Comparison of a non-pathogenic clone A1np with pathogenic clone B2p revealed 76 differentially expressed genes, whereas comparison of a non-pathogenic clone B8np with B2p revealed only 19 differentially expressed genes. Only six genes were found to be similarly regulated in the two non-pathogenic clones A1np and B8np in comparison with the pathogenic clone B2p. Based on these analyses, we chose 20 candidate genes and evaluated their roles in ALA formation using the respective gene-overexpressing transfectants. We conclude that different mechanisms lead to loss of pathogenicity. In total, we identified eight proteins, comprising a metallopeptidase, C2 domain proteins, alcohol dehydrogenases and hypothetical proteins, that affect the pathogenicity of E. histolytica.

Detection of cabbage yellows fungus Fusarium oxysporum f. sp. conglutinans in soil by PCR and real-time PCR

For the specific detection of Fusarium oxysporum f. sp. conglutinans (Foc), we designed a primer set for PCR and a primer–probe set for real-time PCR based on its endopolygalacturonase gene sequence. Using the primer set, we could distinguish Foc from other pathogenic forms and nonpathogenic isolates of F. oxysporum. Moreover, we detected the fungus by real-time PCR using DNA isolated from soil. Sensitivity of real-time PCR was improved 10- to 10,000-fold by adding bovine serum albumin or by performing pre-PCR. Our method can detect Foc in the soil at a density that causes only slight yellows symptom.

Investigation of potential Shiga toxin producing Escherichia coli (STEC) associated with a local foodborne outbreak using multidisciplinary approaches

Shiga toxin producing Escherichia coli (STEC) outbreak is a public health concern as it can potentially cause a variety of clinical manifestations including diarrhea, hemorrhagic colitis and hemolytic uremic syndrome (HUS). However E. coli are generally innocuous commensal organisms, and there is a need to discriminate pathogenic from non-pathogenic isolates rapidly and accurately. In this study, we have used standard culture based methods and advanced molecular approaches to characterize E. coli in food in a local outbreak investigation. We show that the application of DNA based detection methods including real-time PCR and DNA microarray along with a traditional culture method can identify the organism implicated in an outbreak at the strain level for pathogenic potential.

Association of EffectorSix6 with Vascular Wilt Symptoms Caused byFusarium oxysporumon Soybean

The Fusarium oxysporum species complex (FOSC) is a widely distributed group of fungi that includes both pathogenic and nonpathogenic isolates. In a previous study, isolates within the FOSC collected primarily from soybean were assessed for the presence of 12 fungal effector genes. Although none of the assayed genes was significantly associated with wilt symptoms on soybean, the secreted in xylem 6 (Six6) gene was present only in three isolates, which all produced high levels of vascular wilt on soybean. In the current study, a collection of F. oxysporum isolates from soybean roots and F. oxysporum f. sp. phaseoli isolates from common bean was screened for the presence of the Six6 gene. Interestingly, all isolates for which the Six6 amplicon was generated caused wilt symptoms on soybean, and two-thirds of the isolates showed high levels of aggressiveness, indicating a positive association between the presence of the effector gene Six6 and induction of wilt symptoms. The expression profile of the Six6 gene analyzed by quantitative reverse-transcription polymerase chain reaction revealed an enhanced expression for the isolates that caused more severe wilt symptoms on soybean, as established by the greenhouse assay. These findings suggest the suitability of the Six6 gene as a possible locus for pathogenicity-based molecular diagnostics across the various formae speciales.

Molecular characterization of pathogenic and nonpathogenic fowl aviadenovirus serotype 11 isolates.

Fowl aviadenoviruses, many of which are of importance in veterinary medicine, are classified into 5 species. In this study, a pathogenic isolate and a nonpathogenic isolate of fowl aviadenovirus serotype 11 (FAdV-11) of species Fowl aviadenovirus D were characterized. Growth rates were analyzed for the 2 isolates, showing notable differences. The complete genomic sequences of the viruses were fully determined and were analyzed. The genomes of the 2 isolates showed 98.1% sequence identity and revealed 6 nonsynonymous mutations between the Ontario isolates. Two of the 6 mutations were also found in the sequences of recently published pathogenic Chinese fowl aviadenovirus 11 isolates, suggesting potential molecular markers that could be associated with pathogenesis. Deletions were found in the L5 region within the overlapping coding sequences for the 100, 22, and 33 kDa proteins, and these were found in only the nonpathogenic isolates. This molecular pattern was identified in FAdV-9, another nonpathogenic FAdV-D species virus. Furthermore, the tandem repeat regions varied dramatically; the pathogenic isolates contained a reduced number of tandem repeats compared with the nonpathogenic isolates. Lastly, a protein produced early in infection was analyzed using bioinformatics to determine its role in disease. This study highlights several candidate molecular determinants of avian adenovirus genomes related to pathogenicity.

Endophytic bacteria from Datura metel for plant growth promotion and bioprotection against Fusarium wilt in tomato

ABSTRACTNine non-pathogenic bacterial isolates, recovered from Datura metel organs and able to colonise the internal stem tissues of tomato cultivar Rio Grande, were screened for their ability to suppress tomato Fusarium wilt disease caused by Fusarium oxysporum f. sp. lycopersici (FOL), and to enhance plant growth. S33 and S85 isolates tested were found to be the most effective in decreasing Fusarium wilt severity by 94–95% compared to FOL-inoculated and untreated control. A significant enhancement of growth parameters was recorded on tomato plants inoculated or not with FOL. Both isolates were characterised and identified using 16S rDNA sequencing genes as Stenotrophomonas sp. str. S33 (KR818084) and Pseudomonas sp. str. S85 (KR818087). Screened in vitro for their antifungal activity towards FOL, these isolates led to 38.7% and 22.5% decrease in pathogen radial growth and to the formation of an inhibition zone of 12.75 and 8.37 mm respectively. Stenotrophomonas sp. str. S33 and Pseudomonas sp. str. S85 were found to be chitinase-, protease- and pectinase-producing strains but unable to produce hydrogen cyanide. Production of indole-3-acetic acid-like compounds, phosphate solubilising ability and pectinase activity were investigated for elucidating their plant growth-promoting traits and their endophytic colonisation ability.

Genetic variability among Fusarium oxysporum isolates from melon (Cucumis melo) in Qazvin province, Iran

&lt;p&gt;&lt;em&gt;Fusarium&lt;/em&gt; wilt, caused by &lt;em&gt;Fusarium oxysporum&lt;/em&gt; f. sp. &lt;em&gt;melonis&lt;/em&gt; (Fom), the soil-borne fungus that can due to more losses in yield of melon plants. Fusarium is a variable fungi in terms of morphology, symptom production, pathogenicity and wide host range. From 2013 to 2015, 120 isolates of &lt;em&gt;F. oxysporum,&lt;/em&gt; &lt;em&gt;F. acuminatum, F. graminearum, &lt;/em&gt; &lt;em&gt;F. proliferatum&lt;/em&gt; and &lt;em&gt;F. solani&lt;/em&gt; from melon, were collected from disease suspected plants from the field and defined for pathogenicity in our melon plants. Then, a sort of those were elected for phylogenetic analysis. In the beginning, the &lt;em&gt;Fusarium&lt;/em&gt; isolates were classified based on morphology and identifications were authenticated based on sequence data from, EF1α and DNA, and then were used to determine whether &lt;em&gt;Fusarium&lt;/em&gt; variability is relevant to geographic origin and pathogenicity. Neighbor-joining analyses datasets indicated some clades based on geographic origin but there is not even a single clades including solely of pathogens. Because of the factors affecting in pathogenicity are variable, we should be considered them in future studies. Due to the presence of FOM and some nonpathogenic isolates in almost all clades, it is obvious that &lt;em&gt;Fusarium oxysporum&lt;/em&gt; f. sp. &lt;em&gt;melonis&lt;/em&gt; is not monophyletic.&lt;/p&gt;

Co-occurrence of pathogenic and non-pathogenic Fusarium decemcellulare and Lasiodiplodia theobromae isolates in cushion galls disease of cacao (Theobroma cacao L.)

AbstractFlowery cushion gall of cacao is a disease complex with six types. Fusarium decemcellulare have been isolated from both flowery and green point galls and recognized as the etiological agent of the disease. In the present work we: i) identified by ITS-rDNA sequencing and/or taxonomy the cultivable fungal species or Operative Taxonomic Units (OTUs) associated with the five symptoms of cushion galls in cacao from Venezuela, and ii) determined the gall inducing capacity on cacao peeled seeds after 45 days of inoculation with suspensions of mycelia/ spores from distinct isolate types. The whole isolate collection rendered an abundance of 113 isolates with a richness of 39 OTUs (27) and eight identified at the species or genera levels, respectively, and in unidentified fungi. The dominant recovered species (≈36%) were F. decemcellulare and Lasiodiplodia theobromae. Some isolates of F. decemcellulare, L. theobromae, F. equiseti, Fusarium spp., F. solani, F. incarnatum, Rhizocthonia solani and Penicillium sp. were pathogenic. Some other isolates of the first six mentioned taxa behave as non-pathogenic. Furthermore, pathogenic and non-pathogenic isolates can also co-occur within a single plant and gall type. Moreover, 2-5 species within a single gall symptom in a single tree were identified (not necessarily at the same point in the tree), indicating a broad diversity of co-occurring taxa.

Fungal root endophytes of tomato from Kenya and their nematode biocontrol potential

The significance of fungal endophytes in African agriculture, particularly Kenya, has not been well investigated. Therefore, the objective of the present work was isolation, multi-gene phylogenetic characterization and biocontrol assessment of endophytic fungi harbored in tomato roots for nematode infection management. A survey was conducted in five different counties along the central and coastal regions of Kenya to determine the culturable endophytic mycobiota. A total of 76 fungal isolates were obtained and characterized into 40 operational taxonomic units based on the analysis of ITS, β-tubulin and tef1α gene sequence data. Among the fungal isolates recovered, the most prevalent species associated with tomato roots were members of the Fusarium oxysporum and F. solani species complexes. Of the three genes utilized for endophyte characterization, tef1α provided the best resolution. A combination of ITS, β-tubulin and tef1α resulted in a better resolution as compared to single gene analysis. Biotests demonstrated the ability of selected non-pathogenic fungal isolates to successfully reduce nematode penetration and subsequent galling as well as reproduction of the root-knot nematode Meloidogyne incognita. Most Trichoderma asperellum and F. oxysporum species complex isolates reduced root-knot nematode egg densities by 35–46 % as compared to the non-fungal control and other isolates. This study provides first insights into the culturable endophytic mycobiota of tomato roots in Kenya and the potential of some isolates for use against the root-knot nematode M. incognita. The data can serve as a framework for fingerprinting potential beneficial endophytic fungal isolates which are optimized for abiotic and biotic environments and are useful in biocontrol strategies against nematode pests in Kenyan tomato cultivars. This information would therefore provide an alternative or complementary crop protection component.

In vivo virulence of viral haemorrhagic septicaemia virus (VHSV) in rainbow trout Oncorhynchus mykiss correlates inversely with in vitro Mx gene expression

The in vitro replication of viral haemorrhagic septicaemia virus (VHSV) isolates from each VHSV genotype and the associated cellular host Mx gene expression were analysed. All the isolates were able to infect RTG-2 cells and induce increased Mx gene expression (generic assay detecting isoforms 1 and 3 [Mx1/3]). A trout pathogenic, genotype Ia isolate (J167), showing high replication in RTG-2 cells (by infective titre and N gene expression) induced lower Mx1/3 gene expression than observed in VHSV isolates known to be non-pathogenic to rainbow trout: 96-43/8, 96-43/10 (Ib); 1p49, 1p53 (II); and MI03 (IVb). Paired co-inoculation assays were analysed using equal number of plaque forming units per ml (PFU) of J167 (Ia genotype) with other less pathogenic VHSV genotypes. In these co-inoculations, the Mx1/3 gene expression was significantly lower than for the non-pathogenic isolate alone.Of the three rainbow trout Mx isoforms, J167 did not induce Mx1 up-regulation in RTG-2 or RTgill-W1 cells. Co-inoculating isolates resulted in greater inhibition of Mx in both rainbow trout cell lines studied. Up-regulation of sea bream Mx in SAF-1 cells induced by 96-43/8 was also lower in co-inoculation assays with J167. The RTG-P1 cell line, expressing luciferase under the control of the interferon-induced Mx rainbow trout gene promoter, showed low luciferase activity when inoculated with pathogenic strains: J167, DK-5131 (Ic), NO-A-163/68 (Id), TR-206239-1, TR-22207111 (Ie), 99-292 (IVa), and CA-NB00-01 (IVc). Co-inoculation assays showed a J167-dose dependent inhibition of the luciferase activity. The data suggest that virulent VHSV isolates may interfere in the interferon pathways, potentially determining higher pathogenicity.