Strain-dependent effects of environmental signals on the production of extracellular phospholipase by Cryptococcus neoformans

Extracellular phospholipase (PL) activities comprising phospholipase B, lysophospholipase and lysophospholipase transacylase have been identified in culture supernatants of Cryptococcus neoformans and contribute to virulence. We found that PL production was optimal after fungal growth at 30 degrees C and secretion at 37 degrees C for all six C. neoformans isolates studied (four C. neoformans var. neoformans and two C. neoformans var. gattii). No increase in PL activity was found in one strain, NU-2, in low iron or tissue culture media, conditions where upregulation of other virulence factors has been reported. The most virulent strains in an intravenous mouse model of infection were best able to produce PL at growth and secretion temperatures of 37 degrees C, in tissue culture media and under assay conditions of pH 7.0.     

Studies on virulence of the take-all fungus, Gaeumannomyces graminis, with reference to methology

Current methods for take-all assessment in laboratory experiment were examined; it was shown that the extent of vascular discoloration may not reflect virulence of a fungal isolate or host resistance to the pathogen under some experimental conditions. A new assessment method for take-all is described, based on the ability of transport eosin past infection sites. It enables hosts or isolates to be compared by ET₅₀ values, the times from inoculation when 50% of plants fail in eosin-uptake through the three oldest seminal roots. Use of this technique suggested that barley roots were less affected than were wheat roots by Gaeumannomyces graminis var. tritici. Further experimental results showed that an isolate of G. graminis that had lost part of its virulence in culture yielded some single-conidium progeny more virulent than itself. When single-condium isolates or a mycelial isolate and its single-conidium progeny were jointly inoculated on wheat, the amount of disease was less than that caused by the more virulent isolate alone.     

Expression of collagenase in Flavobacterium psychrophilum isolated from cold-water disease-affected ayu (Plecoglossus altivelis)

The collagenase activity and the fpcol gene were examined in Flavobacterium psychrophilum isolates from cold-water disease (CWD)-affected ayu, Plecoglossus altivelis. Collagenase expression was closely related to the accumulated mortality of CWD-affected ayu. RT-qPCR and bacterial challenge experiments showed that F. psychrophilum ayu isolate WA-1 expressed the fpcol gene more actively and was more virulent than ayu isolate WA-2. The amago (Oncorhynchus masou) isolate WB-1, which possesses a pseudo-fpcol gene, was not harmful to ayu. Hitherto, the well-studied metalloproteases Fpp1 and Fpp2 have been considered virulence factors. However, the most virulent isolate against ayu (WA-1) showed no Fpp activity because of a deletion mutation or an insertion of a transposon in the fpp genes. The less virulent WA-2 isolate showed only Fpp1 activity. Taken together, these results suggest that collagenolytic activity, but not Fpp activity, is related to the virulence of F. psychrophilum isolates in CWD-affected ayu.     

Hypovirulence of Didymella bryoniae Associated with dsRNA

Didymella bryoniae, isolate 98–18, recovered from watermelon seedlings with symptoms of gummy stem blight, showed abnormal growth, mycelial lysis, sectoring, barrage and limited production of fruiting bodies in culture. A dsRNA (approximately 6.5 kbp) was associated with isolate 98–18 and other isolates showing abnormal mycelial growth. Transfer of dsRNA from 98–18 to virulent isolates Tk659 and TK671 via hyphal anastamosis was achieved only by consecutive exposure to 98–18, but not to virulent isolates RJ1 and RJ2. Transfer of dsRNA via infiltration through growth media to virulent isolates RJ1, RJ2, TK659 and TK671 was unsuccessful. The disease severity index of isolate 98–18 was reduced 53% on watermelon, 32% on cataloupe and 26% on yellow squash compared with isolate RJ2, suggesting that the presence of the dsRNA is associated with reduced virulence of D. bryoniae isolate 98–18. Zucchini and yellow squash were resistant to both isolates when tested.     

ERIC-PCR-Generated Genomic Fingerprints and Their Relationship with Pathogenic Variability of Xanthomonas campestris pv. punicae, the Incitant of Bacterial Blight of Pomegranate

Bacterial blight caused by Xanthomonas campestris pv. punicae (Xcp) has emerged as a potential threat in pomegranate (Punica granatum) cultivation in India. Here, we report the genomic fingerprints and their correlation with virulence pattern of Xcp isolates from Maharashtra and Delhi. The genomic fingerprints of Xcp isolates were generated using enterobacterial repetitive intergenic consensus (ERIC) sequence-based primers, and virulence level was based on their reaction upon infiltration to susceptible pomegranate cultivar. Maharashtra isolate PGM1 showed only 50% similarity with Delhi isolate PGD8 forming a distinct genotype, whereas the Delhi isolates PGD5 and PGD6 form a cluster with Maharashtra isolates PGM2 and PGM4. The isolates PGM2, PGM4, PGD5, and PGD6 showing mean disease score of 7.47 were marked as group A or highly virulent. The moderately virulent or group B isolates PGM3 and PGD7 produced mean disease score of 4.19, whereas less virulent or group C isolates PGD8 and PGM1 gave mean disease intensity of 1.91. A correlation between genotypic groups based on ERIC fingerprints and pathogenicity of the isolates was established. The highly virulent isolates PGM2, PGM4, PGD5, and PGD6 formed a single cluster. A unique 900 bp amplicon present in all highly virulent isolates has been identified that can be used as genetic marker to screen isolates for virulence. The less virulent isolates PGD8 and PGM1 formed single cluster at 50% similarity coefficient. This seems to be the first report to establish a correlation between ERIC-PCR fingerprints and their corresponding virulence pattern of the pomegranate bacterial blight pathogen.     

Traits associated with virulence to the aphid Macrosiphoniella sanborni in eighteen isolates of Verticillium lecanii

Eighteen isolates of the entomopathogenic fungus Verticillium lecanii from various world-wide locations, from insect hosts and soil were bioassayed against the aphid Macrosiphoniella sanborni in the laboratory. Virulence ranged from an isolate which achieved 100% mortality and LT 50 value (adults) of 3 days ± 0· 2 at 24 ° C, compared with the least virulent isolates causing less than 10% mortality over 14 days (when treated with an inoculum of 1 × 10⁶ conidiospores/ml). All isolates produced extracellular protease and lipase, irrespective of their virulence. A number of traits were frequently associated with the expression of virulence including fast germination, high sporulation rate, an absence of extracellular amylase activity and high extracellular chitinase activities. Large spore size was not strongly associated with virulence. There were exceptions in each variate studied, suggesting that overall expression of virulence is a result of the total complex of these and other traits still to be determined.     

A Possible Proteome-level Explanation for Differences in Virulence of Two Isolates of a Fungal Pathogen Alternaria brassicae

Brassica napus (canola) is an important oilseed crop in many countries throughout the world including Canada. One of the major constraints on canola productivity is blackspot disease caused by the necrotrophic phytopathogenic fungus Alternaria brassicae. Two isolates of A. brassicae with significant differences in virulence have been characterized at the proteomelevel. The morphological observations indicated the Ontario isolate to be more virulent by virtue of increased disease severity score as compared to the UAMH7476 isolate. This was further confirmed through histological observations that indicated extensive colonization of the host tissue by the highly virulent isolate. Mycelial protein profiles of two differentially virulent A. brassicae isolates were compared using two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS) in order to identify proteins that may be responsible for the observed differences. The investigation suggested several differences in the mycelial proteomes of the two isolates. The proteins that were significantly abundant in the more virulent isolate included a protein with conserved actin related protein 2/3 domain, enolase, malate dehydrogenase and serine protease. These results suggest that the differential protein expression pattern could be exploited to identify putative virulence and pathogenicity factors in A. brassicae.     

Vegetative Compatibility Among Isolates of Colletotrichum gloeosporioides from Yam (Dioscorea spp.) in Nigeria

Isolates of Colletotrichum gloeosporioides obtained from yam‐based cropping systems in Nigeria, previously characterized on the basis of morphology, virulence and rDNA internal transcribed spacer (ITS) sequence variation were further compared for vegetative compatibility (VC). Chlorate‐resistant nitrate non‐utilizing (nit) mutants were generated from the isolates and used in complementation (heterokaryon) tests. Tests of VC between complementary mutants from different isolates indicated the presence of several genotypes within a single field, suggesting limited clonal spread. In some cases, isolates obtained from the same lesion were observed to belong to different vegetative compatibility groups (VCGs). No compatibility was observed between isolates of the highly virulent slow‐growing grey (SGG), the moderately virulent fast‐growing salmon (FGS) and the avirulent/weakly virulent fast‐growing grey (FGG) strains. Forty‐one C. gloeosporioides isolates belonged to 28 VCGs, giving a genotype diversity estimate of 0.68. This diversity confirmed the high variability of the pathogen population as revealed by previous characterization studies, however, a correlation between VCGs and isolate groupings based on morphology and virulence was not found. The finding that an isolate from weed was compatible with yam isolates indicated that transfer of important traits, such as virulence, may take place between isolates from yam and non‐yam hosts. The VCG diversity revealed by this study suggests that in addition to asexual reproduction, sexual reproduction may play an important role in the epidemiology of anthracnose on yam.     

Virulence of Septoria nodorum as Affected by Passage Through Detached Leaves of Wheat and Barley Cultivars1

Abstract Three genetically marked, single–spore isolates of Septoria nodorum from wheat were passed through detached leaves of wheat cvs Blueboy and Coker 747 and the barley cv. Boone to produce three sub–isolates per original isolate. Each sub–isolate was cultured for three pycnidiospore generations on its respective host. Virulence of each sub–isolate on detached leaves of Blueboy, Caldwell, Coker 747, and NK81W701 wheat, and Boone and Surry barley was compared with that of the original single–spore isolate from which it was derived. In most cases, sub–isolates passed through wheat were significantly more virulent than the originals on wheat cultivars. They also were more virulent to barley than the original isolates but they were less virulent to barley than to wheat cultivars. Isolate × cultivar interactions were statistically significant (P < .0001) for isolates passed through wheat or barley and were greater than isolate × cultivar interactions among the original isolates. In seven of eight isolates passed through wheat or barley, only the original genetic marker was recovered after three generations, indicating that cross–contamination could not account for the observed change of virulence. In the single case of apparent contamination, of a sub–isolate, virulence declined.     

Un vivo depletion of murine CD8 positive T cells impairs survival during infection with a highly virulent strain ofCryptococcus neoformans

Cell-mediated immunity plays an important but incompletely understood role in host defense againstCryptococcus neoformans. Because of their multiple capacities as cytokine-secreting cells, cytotoxic cells, and antigen-specific suppressor cells, CD8 positive T lymphocytes could potentially either enhance or impair host defense againstC. neoformans. To determine whether CD8 T cells enhance or inhibit host defence during an infection with a highly virulent strain ofC. neoformans, we examined the effect of in vivo CD8 cell depletion on suNival and on the number of organisms in mice infected by either the intratracheal or intravenous routes. Adequacy of depletion was confirmed both phenotypically and functionally. Regardless of the route of infection, we found that survival of mice depleted of CD8 T cells was significantly reduced compared to undepleted mice. Surprisingly, however, CD8 depletion did not alter organism burden measured by quantitative CFU assay in mice infected by either route. These data demonstrate that CD8 positive T cells participate in the immune response to a highly virulent strain ofC. neoformans. By contrast to minimally virulent isolates that do not cause a life threatening infection, the immune response to a highly virulent isolate does not alter the burden of organisms, but does enhance host defense as it is necessary for the optimal survival of infected mice.Abbreviations ³H-TdR ³H-thmidine - CFU colony forming units - FITC Fluorescein isothiocyanate - MLR mixed lymphocyte reaction - PBS phosphate buffered saline     

Characterisation of Isaria fumosorosea isolates and their virulence toward the Diamondback Moth,Plutella xylostella

Some morphological and physiological characteristics of an Isaria fumosorosea isolate with diminished virulence, IFCF01-D, and its parent isolate, IFCF01, were evaluated and laboratory bioassays were performed to assess their virulence against Plutella xylostella. The relationship among these traits and virulence against P. xylostella is discussed. There were no significant differences in conidial viability, spore production and the time required for 50% germination (GT₅₀). Spore viability after incubation for 24 h at 25°C was greater than 98% for both isolates tested. Spore production on potato dextrose agar after 14 days incubation at 25°C was 4.68 × 10⁸ and 4.59 × 10⁸ conidia/mL for IFCF01 and IFCF01-D, respectively. When exposed to high temperatures (40, 45, 50 or 55°C) through a water bath for 10 min, conidial germination ranged from 0.83% to 84.0% for IFCF01 and 0% to 86% for IFCF01-D. Germination rate showed a negative relationship with the exposure temperature for both isolates. The per cent germination of isolate IFCF01 24 h after ultraviolet (UV) radiation (18 W, 240–260 nm) varied from 0% to 92% and 0% to 81% for IFCF01-D. Germination rate and the exposure time exhibited a negative correlation for both isolates tested. Conidial surface hydrophobicity of IFCF01 (60%) was significantly higher than that of isolate IFCF01-D (53%). Subsequently, using the cicada exuviae as the substrate for enzymatic analysis, Pr1 and chitinase activity demonstrated the contrasting virulence traits: higher specific activities for the more virulent IFCF01 and lower enzymatic levels for isolate IFCF01-D.     

Burkholderia pseudomallei virulence: definition, stability and association with clonality.

Clinical presentations of melioidosis, caused by Burkholderia pseudomallei are protean, but the mechanisms underlying development of the different forms of disease remain poorly understood. In murine melioidosis, the level of virulence of B. pseudomallei is important in disease pathogenesis and progression. In this study, we used B. pseudomallei-susceptible BALB/c mice to determine the virulence of a library of clinical and environmental B. pseudomallei isolates from Australia and Papua New Guinea. Among 42 non-arabinose-assimilating (ara(-)) isolates, LD(50) ranged from 10 to > 10(6) CFU. There were numerous correlations between virulence and disease presentation in patients; however, this was not a consistent observation. Virulence did not correlate with isolate origin (i.e. clinical vs environmental), since numerous ara(-) environmental isolates were highly virulent. The least virulent isolate was a soil isolate from Papua New Guinea, which was arabinose assimilating (ara(+)). Stability of B. pseudomallei virulence was investigated by in vivo passage of isolates through mice and repetitive in vitro subculture. Virulence increased following in vivo exposure in only one of eight isolates tested. In vitro subculture on ferric citrate-containing medium caused attenuation of virulence, and this correlated with changes in colony morphology. Pulsed-field gel electrophoresis and randomly amplified polymorphic DNA typing demonstrated that selected epidemiologically related isolates that had variable clinical outcomes and different in vivo virulence were clonal strains. No molecular changes were observed in isolates after in vivo or in vitro exposure despite changes in virulence. These results indicate that virulence of selected B. pseudomallei isolates is variable, being dependent on factors such as iron bioavailability. They also support the importance of other variables such as inoculum size and host risk factors in determining the clinical severity of melioidosis.     

Evaluation of Durum Wheat Genotypes for Resistance against Root Rot Disease Caused by Moroccan Fusarium culmorum Isolates

Fusarium culmorum is one of the most important causal agents of root rot of wheat. In this study, 10 F. culmorum isolates were collected from farms located in five agro-ecological regions of Morocco. These were used to challenge 20 durum wheat genotypes via artificial inoculation of plant roots under controlled conditions. The isolate virulence was determined by three traits (roots browning index, stem browning index, and severity of root rot). An alpha-lattice design with three replicates was used, and the resulting ANOVA revealed a significant (P < 0.01) effect of isolate (I), genotype (G), and G × I interaction. A total of four response types were observed (R, MR, MS, and S) revealing that different genes in both the pathogen and the host were activated in 53% of interactions. Most genotypes were susceptible to eight or more isolates, while the Moroccan cultivar Marouan was reported resistant to three isolates and moderately resistant to three others. Similarly, the Australian breeding line SSD1479-117 was reported resistant to two isolates and moderately resistant to four others. The ICARDA elites Icaverve, Berghisyr, Berghisyr2, Amina, and Icaverve2 were identified as moderately resistant. Principal component analysis based on the genotypes responses defined two major clusters and two sub-clusters for the 10 F. culmorum isolates. Isolate Fc9 collected in Khemis Zemamra was the most virulent while isolate Fc3 collected in Haj-Kaddour was the least virulent. This work provides initial results for the discovery of differential reactions between the durum lines and isolates and the identification of novel sources of resistance.     

Variability in Fusarium oxysporum f. sp. ciceri causing vascular wilt in chickpea

The variability in cultural characteristics and the virulence among three isolates of Fusarium oxysporum f. sp. ciceri causing vascular wilt in chickpea was studied under laboratory conditions. The three isolates (Foc-1, Foc-2 and Foc-3) did not show any significant difference in their mycelial dry weight production at any temperature regimes, pH level or the growth media tested. The radial growth on PDA also did not differ significantly in the three isolates. However, some quantitative differences were noted in colony characters and septations in macroconidia of these isolates. The isolate Foc-1 exhibited dull white, thin and flat hairy growth, spreading out like thread, Foc-2 showed a white fluffy colony with irregular aerial margin, while Foc-3 exhibited a pinkish white, slightly fluffy colony with regular margin. Conidia also differed with regard to septation. Three to six septa were present in Foc-2, while there were 2–3 in isolates Foc-1 and Foc-2. These isolates differed significantly with regard to their virulence on test varieties. Isolate Foc-1 was more virulent that Foc-2 or Foc-3 and produced abundant spores.     

Trypanosoma cruzi: infectivity modulation of a clone after passages through different hosts

Although Trypanosoma cruzi virulence can be modified through passages in vivo or long-term in vitro culture, the mechanisms involved are poorly understood. Here we report modifications in the infectivity of a T. cruzi clone after passages in different hosts without detectable changes in parasite genetic patterns. A clone was obtained from a T. cruzi IIe isolate and showed to be less virulent than the original isolate (p<0.05). This clone was enzymatically similar to the original isolate as shown by multilocus enzyme electrophoresis. Infection of this clone was compared by successive passages in mice and guinea pigs. The mouse-passaged subline became more virulent for both host species compared to the guinea pig-passaged subline (p<0.05). The clone line displayed similar random amplified polymorphic DNA patterns before and after passages in different hosts suggesting that alterations in virulence could be a result of a differential expression of virulence factors.     

Cryptococcus neoformans I. Nonencapsulated Mutants

Seven nonencapsulated mutants of Cryptococcus neoformans were isolated from an encapsulated strain of human origin. Initially, the mutants were avirulent for mice. After several months of subculturing, six of the seven isolates reverted to the encapsulated state and possessed varying degrees of virulence. The results of these experiments suggest that a strong correlation exists between the presence of a capsule and the virulence of C. neoformans.     

In vivo production of A-protein, lipopolysaccharide, iron-regulated outer membrane proteins and 70-kDa serine protease by Aeromonas salmonicida subsp. salmonicida

Using specific immunostaining of Western blots, the in vivo expression of several putative virulence factors of Aeromonas salmonicida subsp. salmonicida was demonstrated in infected muscle tissue of Atlantic salmon and rainbow trout. Three virulent isolates of A. salmonicida were used. One isolate was chosen because in vitro it was apparently a non-producer of the 70-kDa serine protease. Infected furuncle tissue was centrifuged and samples of the pellet and supernatant probed for evidence that the components of interest were bacterial cell-associated or secreted. The A-protein was detected in pelleted furuncle material but not in the supernatant. Lipopolysaccharide, both high and low molecular mass, was present in the pellet but only high molecular mass lipopolysaccharide was detected in the furuncle supernatant. Iron-regulated outer membrane proteins were detected in the furuncle pellet. The 70-kDa serine protease was detected in the furuncle supernatant of both protease-producing strains. However, whilst the protease-deficient isolate was demonstrated to produce low levels of the 70-kDa protease when grown in vitro under iron restricted conditions, none could be detected in vivo.     

Variation in virulence to dry beans, soybeans and maize among isolates of Rhizoctonia solani from beans

The relative susceptibility of dry beans ( Phaseolus vulgaris ), soybeans and maize to anastomosis group 4 isolates of Rhizoctonia solani was determined in greenhouse experiments. Large variations in virulence were found among 30 field isolates. This variation was not due to differential reductions in isolate virulence during axenic culture. There was considerable variation among isolates from within the same field but variability within isolates was small. Twelve of 30 isolates of R. solani were highly virulent to dry beans and soybeans, while the others were of low virulence. Soybeans were more susceptible than dry beans to both pre-emergence mortality and hypocotyl disease. No isolates were highly virulent to maize. The importance of using isolates with a high level of virulence for testing soybean cultivars for resistance to Rhizoctonia disease is stressed.