A DNA Damage Checkpoint Pathway Coordinates the Division of Dikaryotic Cells in the Ink Cap Mushroom Coprinopsis cinerea

The fungal fruiting body or mushroom is a multicellular structure essential for sexual reproduction. It is composed of dikaryotic cells that contain one haploid nucleus from each mating partner sharing the same cytoplasm without undergoing nuclear fusion. In the mushroom, the pileus bears the hymenium, a layer of cells that includes the specialized basidia in which nuclear fusion, meiosis, and sporulation occur. Coprinopsis cinerea is a well-known model fungus used to study developmental processes associated with the formation of the fruiting body. Here we describe that knocking down the expression of Atr1 and Chk1, two kinases shown to be involved in the response to DNA damage in a number of eukaryotic organisms, dramatically impairs the ability to develop fruiting bodies in C. cinerea, as well as other developmental decisions such as sclerotia formation. These developmental defects correlated with the impairment in silenced strains to sustain an appropriated dikaryotic cell cycle. Dikaryotic cells in which chk1 or atr1 genes were silenced displayed a higher level of asynchronous mitosis and as a consequence aberrant cells carrying an unbalanced dose of nuclei. Since fruiting body initiation is dependent on the balanced mating-type regulator doses present in the dikaryon, we believe that the observed developmental defects were a consequence of the impaired cell cycle in the dikaryon. Our results suggest a connection between the DNA damage response cascade, cell cycle regulation, and developmental processes in this fungus.     

Latency of Botrytis cinerea Pers.: Fr. and Biochemical Studies During Growth and Ripening of Two Grape Berry Cultivars, Respectively Susceptible and Resistant to Grey Mould

Latency and development of Botrytis cinerea were assessed under field conditions and after artificial inoculation of two grape varieties, Gamay (susceptible) and Gamaret (resistant). When the percentage of latent Botrytis was the same for both varieties, severity of visible grey mould remained very low in Gamaret berries, while Gamay clusters were destroyed by the disease to a high percentage. Some biochemical parameters were measured in berries, such as constitutive and induced anti‐fungal compounds, polymeric proanthocyanidins and lipid peroxidation products as markers of senescence. Differences were observed in polymeric proanthocyanidins (PPRA) of Gamaret compared with those of Gamay. Concentration and mean degree of polymerization (mDP) of PPRA were always higher in the berries of the resistant variety. The inhibitory effect of Gamaret PPRA on enzyme activity remained until harvest whereas Gamay PPRA lost their inhibitory activity at the beginning of véraison. Based on these results, resistance to B. cinerea seems to be linked to the maintainance of the fungus in its latent form in berries, mainly due to the ability of Gamaret PPRA to inhibit macerating fungal enzyme activities.     

Presence of corazonin in three insect species, and isolation and identification of [His7]corazonin from Schistocerca americana.

An ELISA for corazonin, a cardioactive neuropeptide from the American cockroach, Periplaneta americana, was developed. It was used to isolate corazonin from the cockroach Nauphoeta cinerea, the locust Schistocerca americana, and the hawkmoth Manduca sexta. The peptides from Nauphoeta and Manduca had the same retention times as Periplaneta corazonin, and their amino acid compositions also suggested that these peptides are identical with corazonin. The corazonin-immunoreactive peptide from Schistocerca eluted slightly earlier on HPLC than corazonin, and its structure was determined to be [His⁷]corazonin, or pGlu-Thr-Phe-Gln-Tyr-Ser-His-Gly-Trp-Thr-Asn-amide. These results indicate that corazonin is generally present in insects and that its structure has been well conserved.     

Tomato T2 ribonuclease LE is involved in the response to pathogens

T2 ribonucleases (RNases) are RNA-degrading enzymes that function in various cellular processes, mostly via RNA metabolism. T2 RNase-encoding genes have been identified in various organisms, from bacteria to mammals, and are most diverse in plants. The existence of T2 RNase genes in almost every organism suggests an important biological function that has been conserved through evolution. In plants, T2 RNases are suggested to be involved in phosphate scavenging and recycling, and are implicated in defence responses to pathogens. We investigated the function of the tomato T2 RNase LE, known to be induced by phosphate deficiency and wounding. The possible involvement of LE in pathogen responses was examined. Expression analysis showed LE induction during fungal infection and by stimuli known to be associated with pathogen inoculation, including oxalic acid and hydrogen peroxide. Analysis of LE-suppressed transgenic tomato lines revealed higher susceptibility to oxalic acid, a cell death-inducing factor, compared to the wild type. This elevated sensitivity of LE-suppressed lines was evidenced by visual signs of necrosis, and increased ion leakage and reactive oxygen species levels, indicating acceleration of cell death. Challenge of the LE-suppressed lines with the necrotrophic pathogen Botrytis cinerea resulted in accelerated development of disease symptoms compared to the wild type, associated with suppressed expression of pathogenesis-related marker genes. The results suggest a role for plant endogenous T2 RNases in antifungal activity.     

Effect of membrane integrity on survival competition of Botrytis cinerea upon QoI fungicide pyraclostrobin

Botrytis cinerea, the causal agent of the grey mould disease, developed resistance to multiple fungicides. However, the role of cell membrane in survival competition of B. cinerea upon quinone outside inhibitor (QoI) fungicide has not yet been elucidated. In this paper, the enhancement of cystamine, a transglutaminase inhibitor, on membrane integrity of B. cinerea was determined, and the effect of the enhancement on the sensitivity of B. cinerea to pyraclostrobin was investigated. The results showed that pyraclostrobin inhibited mycelial growth with EC₅₀ as 1.122 and 3.042 μg/ml at 24 and 48 hr, respectively. In the treatment of 5 and 50 μg/ml pyraclostrobin, membrane integrity of B. cinerea was broken, causing high permeability, lipid peroxidation, flocculent and malformed surface with vague septum and abundant agglomerates inside and outside the mycelia. Cystamine even at 50 and 200 μg/ml had little inhibitory effect on mycelial growth. However, in presence of 50 or 200 μg/ml cystamine, the mycelia from pyraclostrobin treatment possessed a significantly reduced leakage, lower MDA content, and a revived fibrous and transparent surface. Meanwhile, SEM images showed that membrane integrity of the mycelia was significantly improved and the agglomerates were dramatically disappeared. Synergy assays further revealed that B. cinerea regained less sensitivity to pyraclostrobin inhibition. In conclusion, membrane integrity controls mycelia sensitivity and is required for survival competition of B. cinerea upon pyraclostrobin.     

Neurohumoral basis of circadian rhythmicity in Nephrops norvegicus (L)

A circadian rhythm of activity is demonstrated in single neurons of Nephrops norvegicus (L.). Eyestalk extracts depress neural and locomotor activity. Entrainment of rhythmicity is achieved by the environmental light cycle, apparently acting through the eye.     

In vitro antifungal activity and mode of action of selected polyphenolic antioxidants on Botrytis cinerea

Six selected antioxidants (catechin, quercetin-3-galactoside, cyanidin-3-glucoside, pelargonidin-3-glucoside, ellagic and gallic acids) were evaluated in vitro for their antifungal activities and mode of action on Botrytis cinerea Pers., one of the most important pathogens of strawberries. Inhibitory effects were found for all the tested antioxidants, but varied at different fungal developmental stages. Catechin and quercetin-3-galactoside showed linear inhibitory effects on germ tube elongation, with the highest suppression ratios of 54.8% and 58.8% respectively. No significant effect was found on spore germination between treatments and control. Gallic acid showed very strong and linear inhibition on spore germination (r = ?0.95), but the effect diminished after spore germination. Cyanidin-3-glucoside and pelargonidin-3-glucoside provided effective control on the fungi as concentrations increased. The arresting effect of ellagic acid on development of B. cinerea was quadratic. Ellagic acid inhibited germ tube elongation and mycelial growth at its highest and lowest concentrations, while no effects were observed at its medium concentration used in this study.     

Biological control of Botrytis cinerea on tomato using naturally occurring fungal antagonists

Abstract

In order to evaluate the potential of naturally occurring filamentous fungi having potential as biocontrol agents effective against grey mould and post-harvest fruit rot caused by Botrytis cinerea on tomato, fungal saprophytes were isolated. They were obtained from leaves, fruits and flowers belonging to different species of cultivated and spontaneous Solanaceous plants collected at the horticultural area of La Plata, Argentina. Of 300 isolates screened for inhibition of B. cinerea using the dual culture technique on agar plate, 12 strains inhibited strongly mycelial growth of the pathogen. Among the antagonists one isolate of Epicoccun nigrum (126), four of Trichoderma harzianum (110, 118, 248 and 252) and four isolates of Fusarium spp. decreased the spore germination of B. cinerea between 30 and 70%. These isolates were probed on tomato fruits to evaluate their biocontrol activity against post-harvest grey mould. In growth chamber tests, E. nigrum (27), F. equiseti (22, 105) and T. harzianum (118, 252) reduced the diameter of fruit lesions by 50 – 90% and were selected for further biocontrol assays of tomato plants in the greenhouse. Although there were not significant differences between the treatments and the control, F. equiseti (105), E. nigrum (27) and T. harzianum (118) reduced by 20, 22 and 22 respectively the disease on whole plants. The targeted application of isolates of E. nigrum, T. harzianum and F. equiseti provides a promising alternative to the use of fungicide spray to control B. cinerea on tomatoes.