Scanning Electron Microscopy of Conidium Formation of Stemphylium vesicarium on Onion Leaves

Onion leaves were inoculated with conidia of Stemphylium vesicarium and the development and morphology of conidiophores and conidia on the leaf surface were examined using scanning electron microscopy. Solitary, but usually fasciculate conidiophores emerged through the epidermis. Hyphae growing on or above the leaf surface also differentiated into conidiophores. Conidiophores were straight or flexuous, simple, smooth or verrucose and cylindrical but enlarged apically at the site of conidiumproduction. Smooth, round, bud-like conidial initials were produced singly at the apex of the verrucose conidiophores. As conidia matured, they became oblong to ovoid and densely verrucose. Once the mature conidium seceded, a small pore was visible at the, apex of the conidiogenous cell. Conidiophores proliferated percurrently at the distal region, forming secondary conidiophores and conidia.     

Relationship between onion thrips (Thrips tabaci) and Stemphylium vesicarium in the development of Stemphylium leaf blight in onion

Stemphylium leaf blight caused by Stemphylium vesicarium and onion thrips (Thrips tabaci) are two common causes of leaf damage in onion production. Onion thrips is known to interact synergistically with pathogens to exacerbate plant disease. However, the potential relationship between onion thrips and Stemphylium leaf blight is unknown. In a series of controlled laboratory and field trials, the relationship between thrips feeding and movement on the development and severity of Stemphylium leaf blight were examined. In laboratory assays, onions (“Avalon” and “Ailsa Craig”) with varying levels of thrips feeding damage were inoculated with S. vesicarium. Pathogen colonisation and leaf dieback were measured after 2 weeks. In pathogen transfer assays, thrips were exposed to S. vesicarium conidia, transferred to onion and leaf disease development was monitored. In field trials, insecticide use was examined as a potential indirect means to reduce Stemphylium leaf blight disease and pathogen colonisation by reducing thrips damage. Results from laboratory trials revealed that a reduction in thrips feeding decreased S. vesicarium colonisation of onion leaves by 2.3–2.9 times, and decreased leaf dieback by 40–50%. Additionally, onion thrips were capable of transferring S. vesicarium conidia to onion plants (albeit at a low frequency of 2–14% of plants inoculated). In field trials, the symptoms and colonisation of Stemphylium leaf blight were reduced by 27 and 17%, respectively with the use of insecticide to control thrips. These results suggest that onion thrips may play a significant role in the development of Stemphylium leaf blight, and thrips control may reduce disease in commercial onion fields.     

Effect of dithiocarbamates on cellulase activity in culture filtrates of Trichoderma reesei

Different concentrations of tetramethylthiuram disulfide (TMTD), sodium dimethyldithiocarbamate (NaDDC), and zinc dimethyldithiocarbamate (ZnDDC) affected the amount of cellulase(s) activity in the culture of Trichoderma reesei. After eight days incubation at 28 degrees C the greatest increase in Avicelase, CMCase, and beta-glucosidase over the control were observed at 0.1 ppm (TMTD) and 0.4 ppm (NaDDC and ZnDCC). There was decrease in the growth in the ZnDDC, but beta-glucosidase activity was reduced considerably. Total protein in the culture filtrate increased with the increase in cellulase(s) activity. No change in pH was observed at eight days incubation but pH increased (not exceeding 5.9) at 12 days incubation.     

Effect of the culture filtrates ofStreptomyces on growth and productivity of wheat plants

Streptomyces olivaceoviridis, S. rimosus andS. rochei proved to possess a high capacity for the production of auxins, gibberellins and cytokinin-like substances, together with substantial levels of α-amylase and proteinase. Grain priming with culture filtrates ofS. olivaceoviridis, S. rimosus orS. rochei appeared to enhance growth vigor and crop yield of wheat plants. In the majority of cases, the culture filtrate ofS. olivaceoviridis appeared to be the most effective in this respect. The present results are discussed in relation to the indirect role played by these bacteria in producing plant growth-regulating substances and their effects on growth and yield of wheat.     

Effects of culture filtrates of Blastomyces dermatitidis on neutrophil locomotion

A potent chemotactic activity for neutrophils is detectable in liquid culture filtrates of Blastomyces dermatitidis. The production of this activity is medium-dependent and culture age-dependent. The highest levels of cytotaxin were produced in filtrates of B. dermatitidis grown in tissue culture medium 199 for three or more weeks. This factor(s) stimulates directed as well as random migration. It functions directly and independently of serum. It is stable at -20 degrees C and 56 degrees C, and has a molecular weight greater than 10000 daltons. These properties define a new microbial chemotactic factor.     

Effects of Host-Selective SV-Toxin from Stemphylium vesicarium, the Cause of Brown Spot of European Pear Plants, on Ultrastructure of Leaf Cells

Brown spot of European pear ( Pyrus communis L.) caused by Stemphylium vesicarium (Wallr.) E. Simmons is a disease of economic importance in fruit-growing areas of southern Europe. The pathogen produces two host-specific SV-toxins (SV-toxin I and II) in culture filtrates, which selectively induce veinal necrosis only on susceptible cultivars. Ultrastructural changes in leaf cells treated with SV-toxin were observed by transmission electron microscopy. Plasma membrane modification specifically occurred at the plasmodesmata of susceptible leaf cells. The plasmalemmal invaginations appeared at both ends of the plasmodesmata 3 h after toxin treatment. Many Golgi vesicles were found in the cytoplasm close to or fused to the invaginated plasma membranes. Polysaccharide-like materials were released from the fused vesicles to extracellular spaces between cell walls and invaginated plasma membranes. Membrane fragments from modified plasma membranes and extended desmotubules from plasmodesmata were also observed at invaginated sites. No changes in ultrastructure were detected in other organelles. These results suggest that the target site of SV-toxin action may be on the plasma membranes of susceptible cells.     

Patterns of airborne conidia of Stemphylium vesicarium, the causal agent of brown spot disease of pears, in relation to weather conditions

Seven-day volumetric spore samplers were installed in pear orchards of northern Italy, in the years between 1993 and 2002, and operated continuously during the development of brown spot epidemics (mid-April–mid-August), caused by Stemphylium vesicarium. Aerial concentration of conidia was recorded at 2 h intervals to study their diurnal and seasonal patterns and the influence of weather conditions. The diurnal periodicity of aerial conidia showed a peak around midday and low counts in the dark. The increase in spore concentration was significantly correlated with the reduction of relative humidity and wetness in early morning, and the increase of wind in late morning and afternoon. Conidia of S. vesicarium became easily airborne to form a regular component of the air-spora in pear orchards, while ascospores were caught only sporadically. Differences between years concerned total spore counts and numbers of peaks (defined as days with more than 30 conidia/m³ air per day). Periods with highest spore counts occurred in late-May to early-June (in 2 years), mid to end of June (5 years), or after mid-July (3 years). There was a significant correlation between spore peaks and days with favourable weather conditions, defined as days with air temperature between 15 and 25°C and high humidity, particularly a wet period longer than 10 h. Occurrence of one or more consecutive days with favourable weather conditions determined an increase in the airborne concentration of conidia, which usually lasted some days and then decreased.     

Seasonal variation of chemical profile of Ruta graveolens extracts and biological activity against Fusarium oxysporum,Fusarium proliferatum and Stemphylium vesicarium

Ruta graveolens L. is a plant of agricultural interest due to its biological activity against phytopathogenic microorganism. This activity has been attributed to the presence of alkaloids, furanocoumarins, terpenes and fatty acids. The accumulation of these compounds is affected by environmental factors. The aim of this work was to analyze the seasonal variation the chemical profile of rue and to evaluate the biological activity against phytopathogenic fungi. Extracts were obtained in January, April, June and September 2017, chemically characterized by GC-MS. The antifungal activity was determined by absorbance, four concentrations were evaluated: 4, 8, 16 and 32 mg/mL. In extracts from the aerial parts, fatty acids were the most abundant group in the January sample (29.7%), and alkaloids in the April and June (27.7%) samples. In extracts from the roots, alkaloids were the most abundant group in the four collections. The highest percentage of growth inhibition on Fusarium and Stemphylium was obtained with the extracts of the January sample. Ruta graveolens can be considered an alternative for the management of phytopathogenic fungi of crops of economic importance.     

Effect of cultural conditions on the presence of a cholera-toxin cross-reactive factor in culture filtrates ofAeromonas hydrophila

Culture filtrates ofAeromonas hydrophila isolate SSU were shown to contain an extracellular product that antigenically cross-reacts with cholera enterotoxin as determined by the enzyme-linked immunosorbent assay (ELISA). We have termed this extracellular product cholera-toxin cross-reactive (CTC) factor until further characterization is accomplished. Various cultural conditions were examined to determine maximum CTC factor production. The results indicate that optimal conditions for the highest levels of extracellular CTC factor produced byA. hydrophila grown in CYE broth occurred when cultures were incubated at 37°C with shaking at 100 rpm. CYE culture filtrates caused fluid accumulation in rabbit ligated intestinal loops. Heating the culture filtrates at 56°C for 10 min resulted in the loss of biological activity and antigenic integrity of the CTC factor as determined by the rabbit ligated intestinal loop assay and theELISA, respectively. The data presented in this study suggest that culture filtrate containing this molecule, or aggregate of molecules, may play a role in the pathogenesis ofA. hydrophila-mediated disease. Further studies including the purification of this factor(s) presently are being conducted in our laboratory.     

Effect of gelling agents and antibiotics on adventitious bud regeneration from In vitro leaves of pear

Summary The effect of the type of gelling agent and of several antibiotics on the adventitious bud regeneration from in vitro leaves was tested on eight pear genotypes. The use of gellan gum (Phytagel™) in the medium instead of agar had a very strong positive effect on the rate of adventitious bud regeneration for all pear genotypes tested in this study. This gelling agent induced faster cell divisions than agar, thus more callus was produced on wound sites and subsequently more buds regenerated. Incubation on gellan gum medium during the first 20 d of bud induction was sufficient to induce a stimulatory effect on regeneration and limited the production of hyperhydric buds. In the prospect of Agrobacterium transformation, the effect of several antibiotics was tested. Cefotaxime (200 mg/l) plus ticarcillin/clavulanic acid (100 mg/l) could be used in the culture medium without affecting the frequency of bud regeneration. The inhibition of bud regeneration was obtained with different kanamycin concentrations according to the gelling agent in the medium. On gellan gum medium, a concentration of 100 mg/l of kanamycin was suitable. These conditions can be recommended for experiments on Agrobacterium-mediated transformation of pear, where bacterial inoculation and presence of antibiotics generally reduce and delay bud regeneration.