Laboratory Inoculation Research Articles

Evaluation of Sinorhizobium meliloti strains and commercial rhizobial inoculants for fenugreek production in Canada

Fenugreek (Trigonella foenum-graecum L.) is a versatile annual legume valued for its nutritional, medicinal, and agricultural uses. Like other legumes, fenugreek can conduct biological nitrogen fixation (BNF). Because fenugreek lacks a dedicated commercial rhizobial inoculant, this study aimed to evaluate the effectiveness of six commercial rhizobial inoculants registered for alfalfa and sweet clover, alongside fifteen Sinorhizobium meliloti strains, on two fenugreek varieties, CDC Canafen and Fenucold. Using laboratory inoculation tests and greenhouse experiments, the symbiotic potential of these products and strains was assessed through nodule formation, BNF, dry matter yield, and the amount of nitrogen fixed. There was variability in symbiotic performance across the strain-variety combinations, with the commercial inoculant RIZOLIQ TOP-Alfalfa and the pure strains USDA1811 and USDA1150 demonstrating superior nodulation, % nitrogen derived from the atmosphere (%Ndfa), and dry matter production. CDC Canafen generally exhibited greater nitrogen fixation compared to Fenucold, and the varieties could fix up to 58% and 47% of their required nitrogen, respectively. The study identifies a registered commercial inoculant that can be used on common fenugreek varieties grown in Canada and shows the potential of other S. meliloti strains for future improvements to inoculant offerings.

Enhanced Resistance to Pokkah Boeng Disease in Sugarcane Through Host-Induced Gene Silencing Targeting FsCYP51 in Fusarium sacchari.

Pokkah boeng disease (PBD), a common and highly destructive disease of sugarcane, is mainly caused by Fusarium sacchari. Breeding sugarcane resistant to PBD is challenging due to the limited availability of immune or highly resistant germplasm resources. Host-induced gene silencing (HIGS) based on RNA interference (RNAi) is a promising disease-control method that offers strong disease-targeting ability with low environmental impact. This study found that silencing either three FsCYP51 genes (FsCYP51A, FsCYP51B and FsCYP51C) simultaneous or two of them (FsCYP51A and FsCYP51C) could inhibit the growth, development, and virulence of F. sacchari. Subsequently, we developed CYP51-HIGS transgenic sugarcane lines using gene-gun genetic transformation and obtained seven lines expressing dsFsCYP51. Both the results of laboratory inoculation assays and field trials indicated that all the seven transgenic lines had significant resistance to PBD. Moreover, in the field trials, the yield losses of transgenic sugarcane due to PBD were reduced compared with those of the control. This is the first report using the HIGS strategy to inhibit PBD infection in sugarcane. This breakthrough provides clear guidelines and practical approaches for the future breeding of sugarcane varieties with strong antifungal resistance.

Effect of inoculation (pre‐ vs. post‐grinding) of black pepper on decimal reduction time of Salmonella spp. and Enterococcus faeciumNRRL B‐2354

AbstractThe laboratory inoculation techniques should ideally mimic the real‐life environment to reliably estimate the decimal reduction time (D‐value) of bacteria for process validation. This study aims at investigating the influence of the inoculation method on the D‐value of Salmonella in black pepper powder. Whole black peppercorns were either inoculated prior to grinding (pre‐grinding procedure) or ground and then inoculated (post‐grinding procedure). The ground black pepper was thermaly treated at 80°C for 0‐30 min. The D80°C values of Salmonella inoculated by pre‐grinding and post‐grinding procedures were 5.5 ± 0.8 and 3.9± 0.3, respectively. Salmonella and E. faecium were significantly (p⟨0.05) more thermally resistant in ground black pepper when inoculated pre‐ rather than post‐grinding. Therefore, inoculation protocol must be considered by spice industries while validating the pasteurization process. E. faecium is a suitable surrogate for Salmonella because of its higher decimal reduction time for both inoculation methods.

Begomovirus Inoculation in Arabidopsis and Cassava.

The use of infectious clones to inoculate plant viruses allows for controlled studies that lead to a better understanding of plant-virus interactions. The main methods used for laboratory inoculation of geminiviruses are agroinoculation and biolistics. We describe how to successfully inoculate geminiviruses, focusing on Arabidopsis as a model plant and cassava as a crop.

Lenticel hydration alters the susceptibility of apple fruit to infection by Phlyctema vagabunda

Bull’s eye rot, caused by Phlyctema vagabunda, is an important postharvest rot of apples. A combination of laboratory and field trials were conducted to examine the relationship between infection and status of apple lenticels. Fruit were hydrated and then inoculated, and climatic factors were compared with inoculations and natural infections in the field. In laboratory trials it was shown that increased lenticel density and size led to more infections. Hydration distended lenticel diameter, and dehydration contracted lenticels. A strong relationship with wind run, leaf wetness and temperature were shown to be important for laboratory inoculations of monthly harvested fruit, natural infections in trap fruit experiments, and monthly field inoculations. It was hypothesised that high wind run (> c. 240 km/day) reduced fruit susceptibility by promoting lenticel closure, and when lenticels were open, temperature limited the formation of an infection stroma. If the stroma did not form, postharvest expression of disease was negligible. Leaf wetness over 28 days was also an important factor for infection, probably because of the effect on micro-crack formation and resealing of lenticels during maturation by components of the cuticle. From these results, pre-harvest application of compounds that close lenticels could provide some control of bull’s eye rot without the need for fungicides.

Co-Inoculation of Aflatoxigenic and Non-Aflatoxigenic Strains of Aspergillus flavus to Assess the Efficacy of Non-Aflatoxigenic Strains in Growth Inhibition and Aflatoxin B1 Reduction

The pre-harvest biocontrol approach currently used includes laboratory inoculations using non-aflatoxigenic strains of Aspergillus flavus. This strategy effectively suppresses the indigenous aflatoxigenic strains and reduces aflatoxin accumulation in sweetcorn. The current in vitro study’s main objective is to determine the diametric growth rates of both Aflatoxin (AF)+ and AF− strains and improve the understanding of competitive relationships among these strains in sweetcorn (Zea mays). Sweetcorn kernels inoculated with AF+ strains only, AF− strains only, and co-inoculated with AF+ + AF− strains were investigated for aflatoxin concentrations. The diametric growth results revealed that growth rates of AF− strains at 25 and 30 °C were much greater than AF+ strains, which was in line with previous studies. The in vitro findings showed that the AKR5− and AKL34− biocontrol strains effectively inhibited the colony propagation and subsequent AFB1 contamination (up to 79%) of AF+ strains. On the other hand, the AKR1− and AKL35− were least effective in reducing AFB1 contents only by 58% and 60%, respectively. There was a significant difference (p < 0.05) in the reduction of AFB1 contents achieved by AF− strains of A. flavus. The findings of the present study indicated the reduction in AFB1 with population expressions of AF+ strains by the AF− strains and supports the notion of competitive exclusion through vigorous development and propagation of the non-aflatoxigenic fungi.

Evaluation of Tepi and Its Surrounding Coffee Collections Against Major Coffee Diseases at Gera and Tepi

Coffee has for centuries played an important role in the Ethiopian economy and represents the main cash crop cultivated by small-scale farmers for social, economic, political and ecological sustainability. The estimated coffee production area in Ethiopia is 561,761 ha. In Ethiopia, coffee berry disease (CBD), coffee wilt disease (CWD) and coffee leaf rust (CLR) are foremost factors in reducing the quantity and quality of coffee in the country. developing disease resistant landrace coffee varieties and boost the yield with their intrinsic cup quality, a multidisciplinary research program has been designed and implemented at Jimma agricultural research center. To meet this objectives, local land race development program of Tepi 88 coffee collections were started in 2015 and 108 coffee accessions were collected and planted in 2016 at Tepi and Gera research sub centre by Jimma Agricultural Research Center. so, 88 coffee accessions were evaluated for their resistance using visual assessment, attached berry test to CBD, visual to scores CLR and laboratory inoculation test to CWD. There were significant differences among the Tepi coffee collections tested for resistance to coffee berry disease. The range was from 0% (T-83/2011) – 98% (T-63/2011) most of Tepi coffee accessions show susceptable reaction for CBD but most of the accessions show resistance reaction at Gera. Coffee collections were also evaluated for their resistance to coffee leaf rust under field condition at Tepi research center. Highly significant difference among coffee cultivars in percent coffee leaf rust severity was observed (Table 1). Coffee leaf rust disease percent severity varied between 0.17% (T-41/2011) – 21.7% (T-11/2011). There existed highly significant difference among cultivars both in percent wilt seedlings death and incubation period). Disease severity and incubation period on Tepi coffee collections varied between 9.33% (T-06/2011) –97.3% (T-21/2011) and 132 -10 days respectively. Based on over all detailed analysis of field evaluation and greenhouse test investigation for major diseases, and their Yield potentials, Nine Tepi coffee accessions were promoted to Variety verification.

Effect of dietary medium-chain fatty acids on nursery pig growth performance, fecal microbial composition, and mitigation properties against porcine epidemic diarrhea virus following storage.

An experiment was conducted to evaluate the effect of dietary medium-chain fatty acid (MCFA) addition on nursery pig growth performance, fecal microbial composition, and mitigation of porcine epidemic diarrhea virus (PEDV) following storage. A total of 360 pigs (DNA 400 × 200, Columbus, NE; initially 6.7 ± 0.07 kg) were randomized to pens (5 pigs per pen) on the day of weaning (approximately 20 d of age), allowed a 6-d acclimation, blocked by BW, and randomized to dietary treatment (9 pens per treatment). All MCFA (Sigma-Aldrich, St. Louis, MO) were guaranteed ≥98% purity, including hexanoic (C6:0), octanoic (C8:0), and decanoic (C10:0) acids. Treatment diets were formulated in 2 phases (7 to 11 and 11 to 23 kg BW) and formulated to meet or exceed NRC requirement estimates. Treatments (n = 8) were a dose response including 0%, 0.25%, 0.5%, 1.0%, and 1.5% added MCFA blend (1:1:1 ratio C6:0, C8:0, and C10:0), as well as treatments with individual additions of 0.5% C6:0, C8:0, or C10:0. Fecal samples were collected from pigs fed control and 1.5% MCFA blend diets on days 0 and 14 and analyzed using 16s rDNA sequencing. Following feed manufacture, feed was stored in bags at barn temperature and humidity for 40 d before laboratory inoculation with PEDV. Subsamples of retained feed were inoculated with PEDV to achieve a titer of 104 TCID50/g and separate sample bottles were analyzed on 0 and 3 d post-inoculation (dpi). Overall, ADG and ADFI were increased (linear, P ≤ 0.010) and feed efficiency (G:F) improved (linear, P = 0.004) with increasing MCFA blend. Pigs fed 0.5% C8:0 had greater (P = 0.038) ADG compared with pigs fed the control diet, and G:F was improved (P ≤ 0.024) when pigs were fed 0.5% C6:0, 0.5% C8:0, or 0.5% C10:0 compared with control. An inclusion level × day interaction was observed (quadratic, P = 0.023), where PEDV Ct values increased (quadratic, P = 0.001) on 0 dpi with increasing levels of MCFA blend inclusion and also increased on 3 dpi (linear, P < 0.001). Fecal microbial diversity and composition were similar between control and 1.5% MCFA blend. In summary, the use of MCFA in nursery pig diets improves growth performance, provides residual mitigation activity against PEDV, and does not significantly alter fecal microbial composition.

Evaluation of 110 Apple Cultivars for Resistance to Alternaria Blotch Caused by Alternaria alternata Apple Pathotype

Apple blotch caused by Alternaria alternata apple pathotype is a severe disease of apple (Malus ×domestica Borkh) occurring throughout the world, especially in eastern Asia. Phenotypic and genetic information about resistance/susceptibility of apple germplasm to this disease will be extremely valuable for selecting and developing new disease resistant cultivars. In this study, 110 apple cultivars obtained from the USDA apple germplasm in Geneva, NY, were evaluated for their resistance/susceptibility to apple blotch by field surveys, and inoculation of detached leaves with a suspension of germinated conidia of A. alternata apple pathotype. Disease incidence were different among the cultivars and categorized into resistant (R), moderately resistant (MR), or susceptible (S). Two molecular markers, S428, a random amplified polymorphic DNA (RAPD) marker associated with disease resistance, and a simple sequence repeat (SSR or microsatellite) marker CH05g07, linked to susceptibility were used to correlate the phenotypes expressed in field surveys and laboratory inoculations. The detection using either the S428 marker or the CH05g07 marker in 50 common breeding cultivars was consistent with R or S traits except for ‘Bisbee’ and ‘Priscilla’. These two cultivars were MR to apple blotch through phenotyping. However, SSR markers were detected, but RAPD markers were not and therefore were considered susceptible. Combined with the record of resistance to fire blight from Germplasm Resources Information Network (GRIN), ‘Dayton’, ‘Mildew Immune Seedling’, ‘Puregold’, and ‘Pumpkin Sweet’ were highly resistant to both diseases and considered as the best choices of parents for stacking resistance to multiple diseases in breeding program.

Survival of Coliform Bacteria in Virgin Olive Oil.

Coliform bacteria consist of both nonpathogen commensal and human opportunistic pathogen species isolated from different habitats like animals, man, vegetables, and water. Olives normally carry natural nonpathogenic epiphytic bacteria, but during growth, harvest, and processing, one of the final products, represented by virgin olive oil, can be contaminated with coliform. Present study showed that coliform bacteria can survive and reproduce in virgin olive oil containing low level of phenolic compounds. The laboratory inoculation trials demonstrated that when the bacterium Escherichia coli, isolated from the olives carposphere, was transferred in olive oil containing high polar phenols content, equal to 372 mg caffeic acid equivalent per kg, the survival was completely inhibited after 15 days of storage. On the contrary, the bacterium reproduced quickly when it was inoculated in virgin olive oil samples containing lower concentration of polar phenols. The SDS-PAGE analysis of the E. coli proteins showed different electrophoretic patterns when the bacterium was inoculated in the virgin olive oil with high phenolic compounds content, confirming the strong interaction between the olive oil phenols content and the bacterial wall proteins. The SEM ultrastructural observations confirmed the presence of a more higher number of damaged microbial cells in virgin olive oil rich of polar phenols. This finding needs further studies since, in an era of antibiotic resistance, the development of new strategies to fight unwanted food bacteria is promising way for the future.

Manipulating host resistance structure reveals impact of pathogen dispersal and environmental heterogeneity on epidemics.

Understanding how variation in hosts, parasites, and the environment shapes patterns of disease is key to predicting ecological and evolutionary outcomes of epidemics. Yet in spatially structured populations, variation in host resistance may be spatially confounded with variation in parasite dispersal and environmental factors that affect disease processes. To tease apart these disease drivers, we paired surveys of natural epidemics with experiments manipulating spatial variation in host susceptibility to infection. We mapped epidemics of the wind-dispersed powdery mildew pathogen Podosphaera plantaginis in five populations of its plant host, Plantago lanceolata. At 15 replicate sites within each population, we deployed groups of healthy potted 'sentinel' plants from five allopatric host lines. By tracking which sentinels became infected in the field and measuring pathogen connectivity and microclimate at those sites, we could test how variation in these factors affected disease when spatial variation in host resistance and soil conditions was minimized. We found that the prevalence and severity of sentinel infection varied over small spatial scales in the field populations, largely due to heterogeneity in pathogen prevalence on wild plants and unmeasured environmental factors. Microclimate was critical for disease spread only at the onset of epidemics, where humidity increased infection risk. Sentinels were more likely to become infected than initially healthy wild plants at a given field site. However, in a follow-up laboratory inoculation study we detected no significant differences between wild and sentinel plant lines in their qualitative susceptibility to pathogen isolates from the field populations, suggesting that primarily non-genetic differences between sentinel and wild hosts drove their differential infection rates in the field. Our study leverages a multi-faceted experimental approach to disentangle important biotic and abiotic drivers of disease patterns within wild populations.

Cryptosporidium parvum decay during air drying and stockpiling of mesophilic anaerobically digested sewage sludge in a simulation experiment and oocyst counts in sludge collected from operational treatment lagoons in Victoria, Australia

The inactivation of Cryptosporidium species oocysts during sewage sludge treatment is important to protect human health when the residual biosolids are applied to agricultural land. Quantifying the decay of Cryptosporidium species during sludge treatment for microbiological assurance purposes is difficult if low numbers are present in wastewater. The rate of decay of Cryptosporidium parvum oocysts during solar/air drying treatment and in sludge stockpiles in temperate environment conditions was simulated in laboratory inoculation experiments using sludge sampled from a mesophilic anaerobic digester. Oocyst numbers were also determined in settled lagoon sludge samples collected from three operational rural wastewater treatment plants (WWTPs). C. parvum oocysts were enumerated by immunomagnetic separation followed by staining with vital dyes and examination by confocal laser scanning microscopy. An air-drying/storage period equivalent to 11 weeks was required for a 1 log10 reduction of viable oocysts inoculated into digested sludge. Oocyst viability in air-dried and stored digested sludge decreased with time, but was independent of sludge desiccation and dry solids (DS) content. No oocysts were detected in sludge samples collected from the anaerobic digester, and the average concentration of oocysts found in settled lagoon sludge from the rural WWTP was 4.6 × 102 oocysts/g DS.

Adaptation to Partial Resistance to Powdery Mildew in the Hop Cultivar Cascade by Podosphaera macularis.

The hop cultivar Cascade has been grown in the Pacific Northwestern U.S. and elsewhere with minimal input for management of powdery mildew (Podosphaera macularis) for nearly 15 years due to the putatively quantitative resistance in this cultivar. While partial resistance is generally thought to be more durable than qualitative resistance, in 2012, powdery mildew was reported on Cascade in Washington State. Field surveys conducted during 2013 to 2016 indicated increasing prevalence of powdery mildew on Cascade, as well as an increasing number of fungicide applications applied to this cultivar in Washington State. Nearly all isolates of P. macularis tested were able to infect Cascade in laboratory inoculations. However, the greatest number of colonies, most conidia produced, and the shortest latent period was only observed with isolates derived originally from Cascade, as compared with other isolates derived from other cultivars. Further, the enhanced aggressiveness of these isolates was only manifested on Cascade and not six other susceptible cultivars, further indicating a specific adaptation to Cascade by the isolates. There was no evidence of a known major R-gene in Cascade, as seven isolates of P. macularis with contrasting virulence all infected Cascade. Among 158 isolates obtained from hop yards planted to Cascade, only two (1.3%) were able to infect the cultivar Nugget, which possesses the resistance factor termed R6, indicating that isolates of P. macularis virulent on Nugget are largely distinct from those adapted to Cascade. Further, race characterization indicated Cascade-adapted isolates of P. macularis were able to overcome R-genes Rb, R3, and R5, but not other known R-genes. Therefore, multiple R-genes and other sources of partial resistance are expected to provide resistance to Cascade-adapted strains of the fungus. Given the plasticity of the powdery mildew fungus, breeding strategies for powdery mildew need to consider the potential for adaptation to both qualitative and partial resistance in the host.

Local adaptation at higher trophic levels: contrasting hyperparasite-pathogen infection dynamics in the field and laboratory.

Predicting and controlling infectious disease epidemics is a major challenge facing the management of agriculture, human and wildlife health. Co‐evolutionarily derived patterns of local adaptation among pathogen populations have the potential to generate variation in disease epidemiology; however, studies of local adaptation in disease systems have mostly focused on interactions between competing pathogens or pathogens and their hosts. In nature, parasites and pathogens are also subject to attack by hyperparasitic natural enemies that can severely impact upon their infection dynamics. However, few studies have investigated whether this interaction varies across combinations of pathogen–hyperparasite strains, and whether this influences hyperparasite incidence in natural pathogen populations. Here, we test whether the association between a hyperparasitic fungus, Ampelomyces, and a single powdery mildew host, Podosphaera plantaginis, varies among genotype combinations, and whether this drives hyperparasite incidence in nature. Laboratory inoculation studies reveal that genotype, genotype × genotype interactions and local adaptation affect hyperparasite infection. However, observations of a natural pathogen metapopulation reveal that spatial rather than genetic factors predict the risk of hyperparasite presence. Our results highlight how sensitive the outcome of biocontrol using hyperparasites is to selection of hyperparasite strains.

Globisporangium oryzicola sp. nov., causing poor seedling establishment of directly seeded rice.

A new species, Globisporangium oryzicola, was isolated from directly seeded rice seedlings, and from soils of paddy fields and an uncultivated field. Despite their different origins, five of the seven isolates studied caused poor seedling establishment of rice in a laboratory inoculation experiment. The species is characterized by oogonia with smooth-walled or sometimes one projection, with one to two antheridia, and aplerotic oospores. Hyphal swellings were rarely observed. Phylogenetic analyses based on the internal transcribed spacer region of the ribosomal RNA gene and mitochondrial cytochrome c oxidase subunit 1 and 2 genes confirmed that the species differed from other Globisporangium species. This novel species is described and illustrated in detail.

Period of susceptibility of almonds to aflatoxin contamination during development in the orchard

Almonds can be contaminated with aflatoxins, produced mainly by Aspergillus flavus and A. parasiticus. Infection can be facilitated by insect injuries during hull split, which begins four to six weeks before harvest. Within this period, it is unknown which kernel stages are most susceptible to aflatoxin contamination. Developing almonds of the Nonpareil cultivar were inoculated weekly with a spore suspension of A. flavus or A. parasiticus for five weeks after hull split in 2013. The almonds were infested with eggs of the lepidopteron navel orangeworm (NOW) (Amyelois transitella) before each spore inoculation. Aflatoxin levels were quantified at harvest using HPLC. Aflatoxin contamination was consistently higher in NOW-damaged kernels, although aflatoxins were also detected in undamaged kernels at each inoculation date. Insect injury is not required for kernel infection but it is a key risk factor for high aflatoxin contamination. Laboratory inoculations were also performed on Nonpareil almond kernels collected during the summers of 2013 and 2015. Aflatoxin levels were significantly lower on dried almonds but the ability to produce aflatoxins was restored when almonds were incubated with high humidity or when the Aspergillus species were inoculated on almond meal agar amended with ground kernels. Therefore, aflatoxins can accumulate in kernels with low aw, should sufficient moisture favors aflatoxin production. In our field experiment, the orchard micro-climate had sufficient humidity to enable aflatoxin production in both damaged and undamaged dried kernels.

The effects of Symbiodinium (Pyrrhophyta) identity on growth, survivorship, and thermal tolerance of newly settled coral recruits.

For many coral species, the obligate association with phylogenetically diverse algal endosymbiont species is dynamic in time and space. Here, we used controlled laboratory inoculations of newly settled, aposymbiotic corals (Orbicella faveolata) with two cultured species of algal symbiont (Symbiodinium microadriaticum and S.minutum) to examine the role of symbiont identity on growth, survivorship, and thermal tolerance of the coral holobiont. We evaluated these data in the context of Symbiodinium photophysiology for 9months post-settlement and also during a 5-d period of elevated temperatures Our data show that recruits that were inoculated with S.minutum grew significantly slower than those inoculated with S.microadriaticum (occasionally co-occurring with S.minutum), but that there was no difference in survivorship of O.faveolata polyps infected with Symbiodinium. However, photophysiological metrics (∆Fv/F'm, the efficiency with which available light is used to drive photosynthesis and α, the maximum light utilization coefficient) were higher in those slower growing recruits containing S.minutum. These findings suggest that light use (i.e., photophysiology) and carbon acquisition by the coral host (i.e., host growth) are decoupled, but did not distinguish the source of this difference. Neither Symbiodinium treatment demonstrated a significant negative effect of a 5-d exposure to temperatures as high as 32°C under low light conditions similar to those measured at settlement habitats.

Disease progression of vector-mediated Grapevine leafroll-associated virus 3 infection of mature plants under commercial vineyard conditions

Grapevine leafroll-associated virus 3 (GLRaV-3) is associated with the economically damaging grapevine leafroll disease, and is transmitted in a semi-persistent manner by several mealybug species. We performed the first controlled field study of vector-mediated inoculations with GLRaV-3 in a commercial vineyard with previously asymptomatic vines, and monitored the vines during four growing seasons. We then compared the outcome of vector-mediated inoculations in the field study to an analogous laboratory study. In the vineyard, about half of all inoculated plants became infected with GLRaV-3, fewer than in the controlled laboratory inoculations. Mealybugs had lower settling and feeding success in the field than in the laboratory inoculations. Our study suggests that laboratory studies may overestimate transmission efficiency. All successfully inoculated vineyard plants first became symptomatic one growing season after inoculations, and berry quality declined within one year after inoculations. Vineyard plants were effective virus sources one year after inoculations. Our findings generally indicate that newly symptomatic vines in commercial vineyards probably became infected during the previous growing season, and a decline in berry quality can be expected during the same year in which symptoms appear.

Pythium kandovanense sp. nov., a fungus-like eukaryotic micro-organism (Stramenopila, Pythiales) isolated from snow-covered ryegrass leaves.

Pythiumkandovanense sp. nov. (ex-type culture CCTU 1813T = OPU 1626T = CBS 139567T) is a novel oomycete species isolated from Lolium perenne with snow rot symptoms in a natural grassland in East-Azarbaijan province, Iran. Phylogenetic analyses based on sequence data from internal transcribed spacer (ITS)-rDNA, coxI and coxII mitochondrial genes clustered our isolates in Pythium group E as a unique, well supported clade. Pythium kandovanense sp. nov. is phylogenetically and morphologically distinct from the other closely related species in this clade, namely Pythium rostratifingens and Pythium rostratum. Pythium kandovanense sp. nov. can be distinguished from these two species by its cylindrical sporangia and lower temperatures for optimum and maximum growth rate. The development of zoospores released through a shorter discharge tube is an additional morphological feature which can be used to differentiate Pythium kandovanense sp. nov. from Pythium rostratifingens. Laboratory inoculation tests demonstrated the pathogenicity of Pythium kandovanense sp. nov. to L. perenne under wet cold (0-3 °C) conditions.

New experimental hosts of Barley yellow dwarf virus among wild grasses, with implications for grassland habitats

Barley yellow dwarf virus (BYDV), an economically important virus, infects small grain cereal crops and over 150 other Poaceae species. BYDV infection plays an important role in competition among grasses in non‐managed systems, but many grasses remain unexamined as potential BYDV hosts. This study examined grass species that have not been reported as BYDV hosts but are commonly encountered in non‐managed grasslands throughout the United States and Canada. Laboratory inoculations with BYDV‐PAV using the aphid vector Rhopalosiphum padi were performed to examine the ability of 13 grass species and barley to be infected with the virus; eight of the grass species were not documented previously as virus hosts. Serological and molecular assays were used to confirm BYDV‐PAV infection. Plant height, number of leaves, number of tillers and weight were recorded to evaluate susceptibility or sensitivity to BYDV. Infection with BYDV was experimentally achieved for the first time on Achnatherum lettermanii, Achnatherum occidentale, Achnatherum thurberianum, Danthonia intermedia, Poa fendleriana, Sporobolus airoides and Sporobolus cryptandrus, but not on Alopecurus pratensis and Elymus wawawaiensis. Infection was confirmed in Bromus inermis, Elymus elymoides, Poa bulbosa, Poa secunda and Hordeum vulgare, which served as controls. BYDV infection caused reductions in plant height on P. bulbosa and P. fendleriana. BYDV‐infected P. secunda had more leaves per plant compared to healthy plants of the same species. BYDV‐infected A. lettermanii exhibited reduced dry weight in both below‐ground and above‐ground tissue. These findings have implications for the management and conservation of grassland habitats.