Infectivity Bioassays Research Articles

Test of Specificity in Signalling between Potato Plants in Response to Infection by Fusarium Solani and Phytophthora Infestans.

Plant-plant signalling via volatile organic compounds (VOCs) in response to insect herbivory has been widely studied, but its occurrence and specificity in response to pathogen attack has received much less attention. To fill this gap, we carried out a greenhouse experiment using two fungal pathogens (Fusarium solani and Phytophthora infestans) to test for specificity in VOC induction and signalling between potato plants (Solanum tuberosum). We paired potato plants in plastic cages, one acting as VOC emitter and the other as receiver, and subjected emitters to one of the following treatments: no infection (control), infected by F. solani, or infected by P. infestans. We measured total emission and composition of VOCs released by emitter plants to test for pathogen-specificity in VOC induction, and then conducted a pathogen infection bioassay to assess resistance levels on receiver plants by subjecting half of the receivers of each emitter treatment to F. solani infection and the other half to P. infestans infection. This allowed us to test for specificity in plant VOC signalling by comparing its effects on conspecific and heterospecific sequential infections. Results showed that infection by neither F. solani or P. infestans produced quantitative (total emissions) or qualitative (compositional) changes in VOC emissions. Mirroring these patterns, emitter infection treatment (control vs. pathogen infection) did not produce a significant change in pathogen infection levels on receiver plants in any case (i.e., either for conspecific or heterospecific sequential infections), indicating a lack of signalling effects which precluded pathogen-based specificity in signalling. We discuss possible mechanisms for lack of pathogen effects on VOC emissions and call for future work testing for pathogen specificity in plant-plant signalling and its implications for plant-pathogen interactions under ecologically relevant scenarios involving infections by multiple pathogens.

Compatibility of Entomopathogenic Nematodes with Chemical Insecticides for the Control of Drosophila suzukii (Diptera: Drosophilidae).

The spotted-wing drosophila, Drosophila suzukii (Matsumura) (Diptera: Drosophilidae), is a pest that reduces the productivity of small fruits. Entomopathogenic nematodes (EPNs) and chemical insecticides can suppress this pest, but the compatibility of the two approaches together requires further examination. This laboratory study evaluated the compatibility of Steinernema brazilense IBCBn 06, S. carpocapsae IBCBn 02, Heterorhabditis amazonensis IBCBn 24, and H. bacteriophora HB with ten chemical insecticides registered for managing D. suzukii pupae. In the first study, most insecticides at the recommended rate did not reduce the viability (% of living infective juveniles (IJs)) of S. braziliense and both Heterorhabditis species. The viability of S. carpocapsae was lowered by exposure to spinetoram, malathion, abamectin, azadirachtin, deltamethrin, lambda-cyhalothrin, malathion, and spinetoram after 48 h. During infectivity bioassays, phosmet was compatible with all the EPNs, causing minimal changes in infectivity (% pupal mortality) and efficiency relative to EPN-only controls, whereas lambda-cyhalothrin generally reduced infectivity of EPNs on D. suzukii pupae the most, with a 53, 75, 57, and 13% reduction in infectivity efficiency among H. bacteriophora, H. amazonensis, S. carpocapsae, and S. brazilense, respectively. The second study compared pupal mortality caused by the two most compatible nematode species and five insecticides in various combinations. Both Heterorhabditis species caused 78-79% mortality among D. suzukii pupae when used alone, and were tested in combination with spinetoram, malathion, azadirachtin, phosmet, or novaluron at a one-quarter rate. Notably, H. bacteriophora caused 79% mortality on D. suzukii pupae when used alone, and 89% mortality when combined with spinetoram, showing an additive effect. Novaluron drastically reduced the number of progeny IJs when combined with H. amazonensis by 270 IJs and H. bacteriophora by 218. Any adult flies that emerged from EPN-insecticide-treated pupae had a shorter lifespan than from untreated pupae. The combined use of Heterorhabditis and compatible chemical insecticides was promising, except for novaluron.

Advancing surgical instrument safety: A screen of oxidative and alkaline prion decontaminants using real-time quaking-induced conversion with prion-coated steel beads as surgical instrument mimetic.

Iatrogenic transmission of prions, the infectious agents of fatal Creutzfeldt-Jakob disease, through inefficiently decontaminated medical instruments remains a critical issue. Harsh chemical treatments are effective, but not suited for routine reprocessing of reusable surgical instruments in medical cleaning and disinfection processes due to material incompatibilities. The identification of mild detergents with activity against prions is therefore of high interest but laborious due to the low throughput of traditional assays measuring prion infectivity. Here, we report the establishment of TESSA (sTainlESs steel-bead Seed Amplification assay), a modified real-time quaking induced cyclic amplification (RT-QuIC) assay that explores the propagation activity of prions with stainless steel beads. TESSA was applied for the screening of about 70 different commercially available and novel formulations and conditions for their prion inactivation efficacy. One hypochlorite-based formulation, two commercially available alkaline formulations and a manual alkaline pre-cleaner were found to be highly effective in inactivating prions under conditions simulating automated washer-disinfector cleaning processes. The efficacy of these formulations was confirmed in vivo in a murine prion infectivity bioassay, yielding a reduction of the prion titer for bead surface adsorbed prions below detectability. Our data suggest that TESSA represents an effective method for a rapid screening of prion-inactivating detergents, and that alkaline and oxidative formulations are promising in reducing the risk of potential iatrogenic prion transmission through insufficiently decontaminated instrument surfaces.

Double trouble: Co‐infection of potato with the causal agents of late and early blight

AbstractGlobal potato production is plagued by multiple pathogens, amongst which are Phytophthora infestans and Alternaria solani, the causal agents of potato late blight and early blight, respectively. Both these pathogens have different lifestyles and are successful pathogens of potato, but despite observations of both pathogens infecting potato simultaneously in field conditions, the tripartite interactions between potato and these two pathogens are so far poorly understood. Here we studied the interaction of A. solani and P. infestans first in vitro and subsequently in planta both in laboratory and field settings. We found that A. solani can inhibit P. infestans in terms of growth in vitro and also infection of potato in both laboratory experiments and in an agriculturally relevant field setting. A. solani had a direct inhibitory effect on P. infestans in vitro and compounds secreted by A. solani had both an inhibitory and disruptive effect on sporangia and mycelium of P. infestans in vitro. In planta infection bioassays revealed that simultaneous co‐inoculation of both pathogens resulted in larger necrotic lesions than single inoculations; however, consecutive inoculations only resulted in larger lesions when A. solani was inoculated after P. infestans. These results indicate that the order in which these pathogens attempt to colonize potato is important for the disease outcome and that the influence of plant pathogens on each other should be accounted for in the design of future disease control strategies in crops such as potato.

A gut-isolated Enterococcus strain (HcM7) triggers the expression of antimicrobial peptides that aid resistance to nucleopolyhedrovirus infection of Hyphantria cunea larvae.

Commensal microorganisms are widely distributed in insect gut tissues and play important roles in to host nutrition, metabolism, reproductive regulation and, especially the immune functioning and tolerance to pathogens. Consequently, gut microbiota represent a promising resource for the development of microbial-based products for pest control and management. However, the interactions among host immunity, entomopathogen infections, and gut microbiota remain poorly understood for many arthropod pests. We previously isolated an Enterococcus strain (HcM7) from Hyphantria cunea larvae guts that increased the survival rates of larvae challenged with nucleopolyhedrovirus (NPV). Here, we further investigated whether this Enterococcus strain stimulates a protective immune response against NPV proliferation. Infection bioassays demonstrated that the re-introduction of the HcM7 strain to germ-free larvae pre-activated the expression of several antimicrobial peptides (particularly H. cunea gloverin 1, HcGlv1), resulting in the significant repression of virus replication in host guts and hemolymph, and consequently improved host survivorship after NPV infection. Furthermore, silencing of the HcGlv1 gene by RNA interference markedly enhanced the deleterious effects of NPV infection, revealing a role of this gut symbiont-induced gene in host defenses against pathogenic infections. These results show that some gut microorganisms can stimulate host immune systems, thereby contributing to resistance to entomopathogens. Furthermore, HcM7, as a functional symbiotic bacteria of H. cunea larvae, may be a potential target for increasing the effectiveness of biocontrol agents against this devastating pest.

Influence of Season and Organic Amendment on the Effectiveness of Different Biosolarization Treatments against Fusarium oxysporum f. sp. lactucae

One strategy presented as an alternative to avoid using chemical substances in soil disinfestation consists in the technique of biosolarization. The aim of the present study was to analyze the effect of seasonality on the effectiveness of biosolarization with different organic amendments for the control of Fusarium oxysporum f. sp. lactucae (FOLAC) on lettuce plants, and to compare the results obtained using a classical soil infectivity bioassay and a qPCR-based molecular technique. None of the plants subjected to biosolarization in the summer season (469–700 and 0–463 h with temperature > 42 °C at 15 and 30 cm soil depth, respectively) showed damage by the pathogen except the untreated control. Conversely, in autumn (3–5 and 0–0 h at temperature = 38–40 °C at 15 and 30 cm soil depth, respectively), only two biosolarization treatments (wheat + semi-composted manure, sunflower pellets) that reduced FOLAC inoculum in soil and plants did not show any disease at the lowest depth (15 cm) in the soil infectivity bioassay. This same result was only obtained at 30 cm soil depth in the biosolarization treatment with sunflower pellets. The number of FOLAC sequences per gram of soil determined with qPCR was null in the biosolarization treatments in summer at both soil depths and corresponded to the absence of disease in the soil infectivity bioassay. A threshold of 145 sequences per gram of soil determined by the qPCR-based molecular technique corresponded to the presence of 10% of diseased lettuce plants infected by FOLAC. Therefore, this molecular technique has been shown to be useful for establishing the soil inoculum thresholds required for crop infection by pathogens, while reducing the time and execution tasks necessary to perform soil infectivity bioassays.

The Potential of a New Beauveria bassiana Isolate for Mosquito Larval Control.

The African malaria mosquito, Anopheles gambiae Giles (Diptera: Culicidae), and the Asian tiger mosquito, Aedes albopictus Skuse (Diptera: Culicidae) are of public health concern due to their ability to transmit disease-causing parasites and pathogens. Current mosquito control strategies to prevent vector-borne diseases rely mainly on the use of chemicals. However, insecticide resistance in mosquito populations necessitates alternative control measures, including biologicals such as entomopathogenic fungi. Here we report the impact of a new Beauveria bassiana (Balsamo) Vuillemin (Hyprocreales: Cordycipitaeceae) isolate, isolated from field-collected Ae. albopictus larvae on mosquito survival and development. Larval infection bioassays using three B. bassiana conidial concentrations were performed on the second and third larval instars of An. gambiae and Ae. albopictus mosquitoes. Larvae were monitored daily for survival and development to pupae and adults. Our results show that B. bassiana MHK was more effective in killing An. gambiae than Ae. albopictus larvae. We further observed delays in development to pupae and adults in both mosquito species exposed the varying concentrations of B. bassiana as compared to the water control. In addition, larval exposure to B. bassiana reduced adult male and female survival in both mosquito species, further contributing to mosquito population control. Thus, this study identifies a new B. bassiana isolate as a possible biological control agent of two mosquito species of public health concern, increasing the arsenal for integrated mosquito control.

Dietary supplementation effect of three microalgae on Penaeus vannamei growth, biochemical composition, and resistance to Vibrio parahaemolyticus (AHPND)

Microalgae are a promising solution in shrimp farming overexploitation. This study assessed the efficacy of microalgae as a food additive for Penaeus vannamei growth and their effect on immune system stimulation against Vibrio parahaemolyticus. Three diets were formulated with 0, 1, and 3% (DC, D1, and D3, respectively) of a mixture of three different microalga species, Tetraselmis suecica, Dunaliella salina, and Chaetoceros muelleri, and growth, survival, and post-harvest quality in shrimp culture were evaluated. Two bioassays were performed: a 60-day feeding trial and an immersion infection bioassay against V. parahaemolyticus. D1 was the best treatment and significantly different to DC (P ≤ 0.05), achieving a food conversion factor of 1.24 ± 0.11 and 1.85 ± 0.38; specific growth rate 1.58 ± 0.03% d-1 and 1.35 ± 0.11% d-1; and weight gain of 5.68 ± 0.32 g and 4.79 ± 0.33 g, respectively. Protein content in shrimp muscle was positively increased by microalgae inclusion, achieving 20.8 ± 0.2% (D1) to 21.7 ± 0.3% (D3), 19.2 ± 0.1% (DC). In the infection bioassay, D1 and D3 reached a 100% survival rate, and histological damage in the hepatopancreas was not observed, suggesting immune system stimulation. These results indicated that microalgae added to food are an excellent source of proteins, carbohydrates, lipids, and promoters of antimicrobial activity that allowed additional protection against mortality caused by V. parahaemolyticus.

Wildfire does not affect spore abundance, species richness, and inoculum potential of arbuscular mycorrhizal fungi (Glomeromycota) in ferruginous Canga ecosystems

Canga ecosystems develop over superficial iron crusts with shallow and nutrient-poor soils. Under these conditions, arbuscular mycorrhizal fungi (AMF) play an important role in helping plants to overcome abiotic and biotic stresses. Canga can suffer periodic burning and yet it is unknown what the impacts of fire are to AMF communities. We aimed to compare AMF in Canga areas affected by burning (BC) with those with no previous history of burning (NC). We compared AMF species composition, spore numbers, species richness, and mycorrhizal inoculum potential. The total number of spores, AMF species richness and mycorrhizal colonization measured in the infectivity bioassay were not significantly different between areas. A total of 23 species in 10 genera were recovered, with most species belonging to Gigasporaceae and Acaulosporaceae. BC and NC shared 52 % of AMF species. Gigaspora albida, Gigaspora gigantea, and Dentiscutata heterogama sporulated exclusively in trap cultures. We concluded that AMF spore communities were not affected by burning in Canga soils as measured by spore abundance, species richness and infectivity. Our data contribute to the inventory of soil biodiversity associated with Canga, a high biodiverse and threatened Brazilian ecosystem.

Pathogen-induced expression of a blight tolerance transgene inAmerican chestnut.

American chestnut (Castanea dentata) is a susceptible host of the invasive necrotrophic fungus Cryphonectria parasitica, which causes chestnut blight disease. The fungal pathogen attacks chestnut stems by invading wounded tissue and secreting oxalate. This process leads to the death of infected host cells and the formation of cankers, eventually girdling stems and killing the tree above the infections. To reduce damage caused by fungal oxalate, American chestnut has been genetically engineered to express a wheat oxalate oxidase (OxO). This enzyme degrades the oxalate produced by the pathogen and confers elevated tolerance to Cryphonectria parasitica infection. We report new lines of transgenic American chestnut that have been developed with the win3.12 inducible promoter from poplar (Populus deltoides) driving OxO expression. This promoter is responsive to both wounding and pathogen infection, with a low level of baseline expression. Targeted expression of OxO to wounded and infected tissue is sought as an alternative to constitutive expression for potential metabolic resource conservation and transgene stability over the long lifetime of a tree and over successive generations of breeding. Transgenic Castanea dentata lines harbouring the win3.12‐OxO construct were evaluated for transgene expression patterns and tolerance to chestnut blight infection. OxO transcript levels were low in uninfected plants, but robust infection‐induced expression levels were observed, with one transgenic line reaching levels comparable to those of previously characterized CaMV35S‐OxO lines. In chestnut blight infection bioassays, win3.12‐OxO lines showed elevated disease tolerance similar to blight‐resistant Chinese chestnut (Castanea mollissima) controls.

Pathogenicity against hemipteran vector insects of a novel insect pathogenic fungus from Entomophthorales (Pandora sp. nov. inedit.) with potential for biological control

A new but still unpublished entomopathogenic fungus (ARSEF13372) in the genus Pandora (Entomophthorales: Entomophthoraceae) was originally isolated from Cacopsylla sp. (Hemiptera: Psyllidae). Several species of the genus Cacopsylla vector phloem-borne bacteria of the genus ‘Candidatus Phytoplasma‘, which cause diseases in fruit crops such as apple proliferation, pear decline and European stone fruit yellows. To determine Pandora’s host range and biocontrol potential we conducted laboratory infection bioassays; Hemipteran phloem-feeding insects were exposed to conidia actively discharged from in vitro produced mycelial mats of standardized area. We documented the pathogenicity of Pandora sp. nov. to species of the insect families Psyllidae and Triozidae, namely Cacopsyllapyri L., C.pyricola (Foerster), C.picta (Foerster, 1848), C.pruni (Scopoli), C.peregrina (Foerster), and Trioza apicalis Foerster. The occurrence of postmortem signs of infection on cadavers within 10 days post inoculation proved that Pandora sp. nov. was infective to the tested insect species under laboratory conditions and significantly reduced mean survival time for C.pyri (summer form and nymph), C.pyricola, C.picta, C.pruni, C.peregrina and T.apicalis. Assessing a potential interaction between phytoplasma, fungus and insect host revealed that phytoplasma infection (‘Candidatus Phytoplasma mali’) of the vector C.picta and/or its host plant apple Malus domestica Borkh. did not significantly impact the survival of C.picta after Pandora sp. nov. infection. The results from infection bioassays were discussed in relation to Pandora sp. nov. host range and its suitability as biocontrol agent in integrated pest management strategies of psyllid pests, including vector species, in orchards.

Prion disease is accelerated in mice lacking stress-induced heat shock protein 70 (HSP70)

Prion diseases are a group of incurable neurodegenerative disorders that affect humans and animals via infection with proteinaceous particles called prions. Prions are composed of PrPSc, a misfolded version of the cellular prion protein (PrPC). During disease progression, PrPSc replicates by interacting with PrPC and inducing its conversion to PrPSc As PrPSc accumulates, cellular stress mechanisms are activated to maintain cellular proteostasis, including increased protein chaperone levels. However, the exact roles of several of these chaperones remain unclear. Here, using various methodologies to monitor prion replication (i.e. protein misfolding cyclic amplification and cellular and animal infectivity bioassays), we studied the potential role of the molecular chaperone heat shock protein 70 (HSP70) in prion replication in vitro and in vivo Our results indicated that pharmacological induction of the heat shock response in cells chronically infected with prions significantly decreased PrPSc accumulation. We also found that HSP70 alters prion replication in vitro More importantly, prion infection of mice lacking the genes encoding stress-induced HSP70 exhibited accelerated prion disease progression compared with WT mice. In parallel with HSP70 being known to respond to endogenous and exogenous stressors such as heat, infection, toxicants, and ischemia, our results indicate that HSP70 may also play an important role in suppressing or delaying prion disease progression, opening opportunities for therapeutic intervention.

An Improved Evans Blue Staining Method for Consistent, Accurate Assessment of Plasmodiophora brassicae Resting Spore Viability.

Clubroot caused by Plasmodiophora brassicae is an important disease of brassica crops. The use of vital stains to determine the viability of P. brassicae resting spores can provide useful information regarding spore longevity, inoculum potential, or the efficacy of antimicrobial treatments. Evans blue is one example of a vital stain that has been reported to differentially stain viable and nonviable resting spores. Some previously published protocols using Evans blue to stain P. brassicae resting spores have not provided accurate or consistent results. In this study, we modified the Evans blue method by increasing the staining time to 8 h or more and evaluated P. brassicae resting spores after heat treatment at various combinations of temperature and time. Extending staining times significantly increased the numbers of stained resting spores up to 7 h, after which the numbers of stained spores did not change significantly (R2 = 96.88; P ≤ 0.001). The accuracy of the modified method to discriminate viable and nonviable spores was evaluated in repeated experiments and by comparing the staining data with those derived from inoculation assays and propidium monoazide quantitative PCR (qPCR). The results demonstrated that the modified Evans blue staining method improved the accuracy and consistency of measurement of P. brassicae resting spore viability. Additionally, it was equivalent to the qPCR method for differentiating viable and nonviable spores (R2 = 99.84; P ≤ 0.001) and confirmed in canola infection bioassays.

Winter biodisinfestation with Brassica green manure is a promising management strategy for Phytophthora capsici control of protected pepper crops in humid temperate climate regions of northern Spain

Phytophthora capsici causes root and crown rot of protected pepper (Capsicum annuum L.) crops in the Basque Country (northern Spain), a humid temperate climate area. The objective was to determine the effect of winter biofumigation and plastic cover (biodisinfestation) with Sinapis alba L. (cultivar 'Ludique') fresh green manure on the survival of introduced P. capsici inoculum (oospores) in a greenhouse soil. After autumn-winter Brassica cover crop soil incorporation in February, oospores remained at 15 and 30 cm depth for four weeks´ time exposure in two consecutive years, 2009 and 2010. Oospores viability was estimated with a plasmolysis method and infectivity with a potted pepper bioassay. Viability was significantly higher in biodisinfestated oospores than in the non-treated control in both years (81% and 21% relative increase in 2009 and 2010, respectively) and significantly higher at 30 cm depth than at 15 cm in 2009 (24% relative increase). Conversely, biodisinfestation significantly delayed disease incidence progression until the first half of infectivity bioassays in both years compared to the non-treated soils (83% and 75% relative decrease of dead plants in 2009 and 2010 respectively). The low soil temperatures could explain the low oospore survival reduction and suggest that the suppressiveness expressed in the infectivity bioassay was related with an increase of microbial activity in the biodisinfestation treatment. We conclude that winter biodisinfestation with fresh Brassica green manure is a promising management strategy for Phytophthora root rot control of protected peppers crops in regions of humid temperate climate such us northern Spain.

The secretome of honey bee-specific lactic acid bacteria inhibits Paenibacillus larvae growth

American Foulbrood (AFB) is a particularly pernicious bacterial disease of honey bees due to the extreme persistence of endospores of the causative agent Paenibacillus larvae. These spores are resistant to harsh environmental conditions, unaffected by antimicrobial agents and can remain viable for decades. The germination of the endospore in the larval midgut is the crucial first step leading to infection, followed by vegetative growth, tissue invasion and disease, culminating in spore formation when the host´s nutrients have been exhausted. Therefore, inhibiting spore germination or impeding early vegetative growth would be a highly effective strategy for limiting the impact of AFB. We previously showed that honey bee-specific lactic acid bacteria (hbs–LAB) had a major inhibitory effect on P. larvae both in culture and in larval bioassays. The present study documents the progress towards characterization of compounds, processes and interactions between P. larvae and the hbs–LAB responsible for this inhibitory effect. Firstly, we used an agar diffusion assay and larval infection bioassay to show that most, if not all, of the inhibitory effect was associated with the extracellular fraction (secretome). Secondly, we employed a turbidimetric growth assay to demonstrate that the hbs–LAB secretome strongly inhibited P. larvae vegetative growth, however, probably not by reducing spore germination. The inhibition was similarly effective against both major P. larvae genotypes (ERIC-I and II) in all experiments. The implications of our results for characterization of the secretome and for the management and treatment of AFB and P. larvae are further discussed.

The symbiotic bacteria Alcaligenes faecalis of the entomopathogenic nematodes Oscheius spp. exhibit potential biocontrol of plant- and entomopathogenic fungi.

SummarySoil‐dwelling entomopathogenic nematodes (EPNs) kill arthropod hosts by injecting their symbiotic bacteria into the host hemolymph and feed on the bacteria and the tissue of the dying host for several generations cycles until the arthropod cadaver is completely depleted. The EPN–bacteria–arthropod cadaver complex represents a rich energy source for the surrounding opportunistic soil fungal biota and other competitors. We hypothesized that EPNs need to protect their food source until depletion and that the EPN symbiotic bacteria produce volatile and non‐volatile exudations that deter different soil fungal groups in the soil. We isolated the symbiotic bacteria species (Alcaligenes faecalis) from the EPN Oscheius spp. and ran infectivity bioassays against entomopathogenic fungi (EPF) as well as against plant pathogenic fungi (PPF). We found that both volatile and non‐volatile symbiotic bacterial exudations had negative effects on both EPF and PPF. Such deterrent function on functionally different fungal strains suggests a common mode of action of A. faecalis bacterial exudates, which has the potential to influence the structure of soil microbial communities, and could be integrated into pest management programs for increasing crop protection against fungal pathogens.

Gene network mediated by WRKY13 to regulate resistance against sheath infecting fungi in rice (Oryza sativa L.)

OsWRKY13 TF gene is known to play a regulatory role of signaling in physiological pathways related to either development or disease resistance in rice plants. Rice cultivars IR 50 and TRY 3, resistant and susceptible respectively to sheath blight, TRY 3 and CO 43 resistant and susceptible respectively to sheath rot were challenged with fungal pathogens and disease scoring was carried out. Percent Disease Index (PDI) was significantly higher in susceptible varieties than resistant varieties. RT-PCR and qPCR analyses of WRKY13 using RNA extracted from the plant tissues revealed higher WRKY13 expression in resistant varieties (both diseases) upon pathogen challenge compared to uninfected control and also the susceptible varieties. To compute and evaluate the possible molecular mechanism for observed resistance correlated to WRKY13 gene expression, rice gene expression profiles against bacterial leaf blight and leaf blast disease from ROAD database were used to prioritize the locus IDs that were used as input in RiceNet v2 tool. The expression of WRKY13-regulated TIFY9 gene was predicted and validated using RT-PCR and qRT-PCR along with WRKY12 and PR2. All three genes showed induced expression in R. solani challenged sheath blight resistant variety. WRKY12 and PR2 expression in S. oryzae challenged sheath rot resistant variety was higher. Agrobacterium mediated transformation was carried out in rice plants using overexpression construct of WRKY13 (agroinfection in seeds of varieties susceptible to sheath blight and sheath rot, followed by selection in antibiotic media, germinating and hardening of putative transgenic lines). Based on qPCR analysis, the expression level of WRKY13 and the co-expression levels of WRKY12, TIFY9 and PR2 were found higher in PCR-positive T1 plants compared to wild-type. Infection bioassays in the transgenic plants of both varieties revealed enhanced resistance to the pathogens. A mechanism by which WRKY13 would influence the MAPK cascade with TIFY9 acting as a mediator, is proposed.

Differential Rickettsial Transcription in Bloodfeeding and Non-Bloodfeeding Arthropod Hosts.

Crucial factors influencing the epidemiology of Rickettsia felis rickettsiosis include pathogenesis and transmission. Detection of R. felis DNA in a number of arthropod species has been reported, with characterized isolates, R. felis strain LSU and strain LSU-Lb, generated from the cat flea, Ctenocephalides felis, and the non-hematophagous booklouse, Liposcelis bostrychophila, respectively. While it is realized that strain influence on host biology varies, the rickettsial response to these distinct host environments remained undefined. To identify a panel of potential rickettsial transmission determinants in the cat flea, the transcriptional profile for these two strains of R. felis were compared in their arthropod hosts using RNAseq. Rickettsial genes with increased transcription in the flea as compared to the booklouse were identified. Genes previously associated with bacterial virulence including LPS biosynthesis, Type IV secretion system, ABC transporters, and a toxin-antitoxin system were selected for further study. Transcription of putative virulence-associated genes was determined in a flea infection bioassay for both strains of R. felis. A host-dependent transcriptional profile during bloodfeeding, specifically, an increased expression of selected transcripts in newly infected cat fleas and flea feces was detected when compared to arthropod cell culture and incubation in vertebrate blood. Together, these studies have identified novel, host-dependent rickettsial factors that likely contribute to successful horizontal transmission by bloodfeeding arthropods.

Phenotypic and Molecular Characterization of Verticillium dahliae, the Causal Agent of Verticillium Wilt of Olive in Tunisia

Aims: During the last two decades, verticillium wilt of olive has spread to young olive orchards were it causes severe yield losses and death of olive trees in southern and central regions of Tunisia. Therefore, identification of the causal agent as well as the study of its pathogenicity will be useful for design the appropriate management program. Place and Duration of Study: This work was performed in the Laboratory of Phytopathology at the Olive Tree Institute (Sfax, Tunisia) between July 2014 and June 2015. Original Research Article Gharbi et al.; JALSI, 4(3): 1-8, 2016; Article no.JALSI.23696 2 Methodology: This study was conducted using phenotypic and molecular methods to identify the causal agent of the decline and death of olive trees. The pathogen was recovered from infested tissues using a potato dextrose agar medium. Identity of the isolates was confirmed by ITS-RFLP and sequencing. Pathogenicity of the isolates was evaluated by infection bioassay on young olive plants. Results: All the fungal isolates were hyaline, flocculose and produced microsclerotia after 15 days of incubation at 25°C, which is in agreement with the identification key of Verticillium species. All the isolates were characterized by ITS-RFLP and sequencing of the internal transcribed spacer. All the isolates produced 470 bp using primers ITS1/ITS4. Digestion of the ITS product using EcoRI and HaeIII produced two fragment of 250 and 220 bp and three fragment of 300, 150, and 20 bp respectively. Pathogenicity was evaluated on two-year old olive plants using an artificial infection bioassay. After 15 days of inoculation, similar symptoms were produced as natural infection. Symptoms of wilt developed rapidly and caused the death of more than 50% of the inoculated plants. Conclusion: V. dahliae is present in almost all growing olive regions of Tunisia. However, local V. dahliae populations in Tunisia is predominated by a highly pathogenic clone which is able to overcome the resistance of the main olive cultivar.

Blue crabs Callinectes sapidus as potential biological reservoirs for white spot syndrome virus (WSSV).

White spot syndrome virus (WSSV) is a virulent pathogen of cultured shrimp and was first detected in farms in South Carolina (USA) in 1997 and subsequently in wild shrimp in 1999. We screened groups of 1808 wild Atlantic white shrimp Litopenaeus setiferus and 300 blue crabs Callinectes sapidus collected from South Carolina, Georgia, and Florida for the presence of WSSV using the Shrimple® immunoassay-strip test, with all positives and random subsets of negatives tested by TaqMan real-time PCR and in infectivity bioassays. Of 87 shrimp and 11 crabs that tested positive using the Shrimple® test, only a single C. sapidus was confirmed to be infected with WSSV by PCR and the infectivity bioassay. The data indicate that the prevalence of WSSV in these species is low in these southeastern US regions, but that C. sapidus may serve as a biological reservoir.