Induction of Resistance to Penicillium digitatum in Grapefruit by β-Aminobutyric Acid

-Aminobutyric acid (BABA), an inducer of pathogen resistance in plants, induced disease resistance in reproductive parts of the plant, such as grapefruit peel tissue. Application of BABA to specific wound sites on the fruit peel surface induced resistance to Penicillium digitatum, the main postharvest pathogen of citrus fruit, in a concentration-dependent manner, being most effective at 20mM, and rather less effective at either higher or lower concentrations. The effect of BABA in inducing resistance to P. digitatum in the fruit peel surface was local and limited to the vicinity (within 1–2cm) of the BABA-treated site. In addition to inducing pathogen resistance, increasing concentrations of BABA (from 1 to 100mM) also exhibited direct antifungal activity and inhibited P. digitatum spore germination and germ tube elongation in vitro. The induction of resistance to P. digitatum by BABA was accompanied by the activation of various pathogen defense responses in grapefruit peel tissue, including activation of chitinase gene expression and protein accumulation after 48h, and an increase in phenylalanine ammonia lyase (PAL) activity after 72h.     

Induction of Chitinase and β-1,3-Endoglucanase Proteins by UV Irradiation and Wounding in Grapefruit Peel Tissue

UV irradiation enhanced the resistance of grapefruit against the development of green mold décay caused byPenicillium digitatum, the main postharvest pathogen of citrus fruit, and significantly inhibited the fungus’ growth at the fruit wound sites. Immunoblotting analysis using specific citrus chitinase and β-1,3-endoglucanase antibodies, showed that UV irradiation, wounding of the fruit, or a combination of these two treatments, induced the accumulation of a 25 kD chitinase protein in the fruit’s peel tissue. On the other hand, UV irradiation or wounding of the fruit alone was unable to induce the accumulation of 39 and 43 kD β-1,3-endoglucanase proteins, but the combination of the two treatments increased these protein levels. It is suggested that both chitinase and β-1,3-endoglucanase may play a role in the UV-induced resistance of grapefruit againstP. digitatum. Contribution from the Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel. No. 403/99. http://www.phytoparasitica.org posting June 3, 1999.     

Immunocytochemical localization of β-1,3-glucanase and chitinase in Fusarium culmorum -infected wheat spikes

Two antisera raised against acidic β-1,3-glucanase and acidic chitinase from tobacco were used to investigate the subcellular localization of the two enzymes in Fusarium culmorum -infected wheat spike by means of the immunogold labelling technique. The studies demonstrated that the distribution of β-1, 3-glucanase and chitinase were very similar in the uninoculated healthy and infected wheat spikes. The enzymes were localized mainly in the cell walls of different tissues including the lemma, ovary and rachis of the wheat spike, while the cytoplasm and organelles of cells in these tissues showed almost no labelling. However, the accumulation of β-1,3-glucanase and chitinase in the infected wheat spikes differed distinctly between resistant and susceptible wheat cultivars. The labelling densities for the two enzymes in the infected lemma, ovary and rachis of the susceptible cultivar Agent increased only slightly as compared to the corresponding uninoculated healthy tissues, whereas higher labelling densities of β-1,3-glucanase and chitinase were found in the infected tissues of wheat spikes from the resistant cultivar Arina compared to the corresponding uninoculated healthy tissues. Furthermore, the labelling of β-1,3-glucanase and chitinase also occurred over the cell walls of the hyphae in the infected wheat spike, but not over the hyphal cytoplasm. In addition, labelling for the two enzymes was often detected over the cell wall appositions and the electron-dense material located between the host cell and the hyphal cell in the infected tissues of the resistant wheat cultivar. The findings reported in the present study indicate that β-1,3-glucanase and chitinase accumulation in the F. culmorum -infected wheat spike may be involved in resistance to pathogen spread in the host tissue.     

Induction of different chitinase andΒ-1,3-glucanase isoenzymes in sunflower (Helianthus annuus L.) seedlings in response to infection byPlasmopara halstedii

Chitinase and-1,3-glucanase activities were assayed in roots, hypocotyls and cotyledons of downy mildewsusceptible and -resistant sunflower (Helianthus annuus L.) cultivars. While the highest-1,3-glucanase activity was in roots, that of chitinase activity was in hypocotyls. Inoculation of both sunflower cultivars withPlasmopara halstedii resulted in a marked increase of chitinase and-1,3-glucanase activities. The increase was observed earlier in incompatible than in compatible reactions. Both enzymes occurred in root tissue as a complex mixture of isoenzymes. At least three different peaks with chitinase activity and three with glucanase activity could be resolved by gel filtration chromatography on Sephacryl S-100 and chromatofocusing on PBE 94 (pH 7-4). Following ammonium sulfate precipitation and ion-exchange on CM- and DEAE-Trisacryl, three glucanase and chitinase fractions, referred to as basic, neutral and acidic, were separated on the basis of their Chromatographic behaviour. A different pattern of distribution of chitinase and-1,3-glucanase fractions was observed between inoculated and non-inoculated plants in both resistant (cv. RS-105) and susceptible (cv. Peredovik) cultivars. In healthy plants-1,3-glucanase was mainly found in the basic (cv. Peredovik) and neutral (cv. RS-105) fractions, whereas chitinase was in the basic fraction for both cultivars. The neutral and acidic fractions of chitinases were induced in the compatible and incompatible reactions. Inoculation of the plants induced the neutral-1,3-glucanase fraction in resistant and susceptible cultivars and the acidic only in the susceptible one. Induction of the basic fraction of both activities was not observed in any case.     

MALDI-Qq-TOF-MS and transient gene expression analysis indicated co-enhancement of β-1,3-glucanase and endochitinase by tMEK2 and the involvement of divergent pathways

A mitogen-activated protein kinase (MAPK) pathway has been demonstrated as a key pathway in plant defense against pathogen attacks. With proteomics approaches, we specifically studied activation events downstream of a MAPK kinase, tMEK2, in tomato. Overexpression of a constitutively activated tomato MAPK kinase gene (tMEK2^MUT) enhanced resistance of transgenic tomato lines to the virulent bacterial pathogen Pseudomonas syringae pv. tomato. Pathogenesis-related genes, PR1b1, β-1,3-glucanase, and endochitinase were up-regulated by tMEK2^MUT. Two-dimensional electrophoresis and matrix-assisted laser desorption/ionisation-time-of-flight-mass spectrometry analysis of total soluble leaf proteins indicated that β-1,3-glucanase and endochitinase are among the up-regulated proteins in these transgenic plants. Co-expression studies using a transient gene expression system have indicated that β-1,3-glucanase and endochitinase genes up-regulated by tMEK2^MUT were down-regulated by different specific phosphatases through dephosphorylation of certain downstream signaling molecules. Our observations indicate that increased products of β-1,3-glucanase and endochitinase genes downstream of tMEK2 may play an important role in achieving disease resistance.     

Comparative resistance to foliar fungal pathogens in transgenic carrot plants expressing genes encoding for chitinase, β-1,3-glucanase and peroxidise

Genes encoding an acidic wheat class IV chitinase (383), an acidic wheat β 1,3-glucanase (638) and a rice cationic peroxidase (POC1) were introduced into ‘Nantes Coreless’ carrot (Daucus carota) by Agrobacterium-mediated transformation. The genes were introduced singly or in various combinations followed by selection imposed by the herbicide phosphinothricin. Regenerated plantlets were screened for presence and expression of the three transgenes using PCR, Southern and Northern hybridisations. Eighteen transgenic lines expressing a single transgene and 2 lines each co-expressing 638/383 and 383/POC1 were assessed for resistance to the necrotrophic fungal pathogens Botrytis cinerea and Sclerotinia sclerotiorum. Percentage leaf area diseased was measured 4 and 7 days after inoculation (dai) and compared to non-transformed control plants. Six lines expressing β-1,3-glucanase 638 alone had no enhanced resistance to B. cinerea at 4 dai and only slight resistance to S. sclerotiorum; there was no effect at 7 dai. Two out of the six lines expressing 383 alone had enhanced tolerance to both pathogens with a 20–50% reduction in disease development at 7 dai. Two lines co-expressing 638/383 had slight reductions in disease by (10–20%) similar to that of the lines expressing chitinase 383 alone. Highest levels of disease resistance were seen in transgenic lines expressing POC1, alone or in combination with chitinase 383. Disease symptoms were slower to develop and symptoms were reduced by up to 90% for B. cinerea and 70% for S. sclerotiorum. The 383/POC1 co-expressing plants developed disease at levels similar to that of POC1 alone. Petioles of plants over-expressing POC1 had higher levels of lignin accumulation constitutively compared to control plants, which was greatly enhanced following inoculation with S. sclerotiorum. These results indicate that peroxidase over-expression can lead to significant disease reduction against necrotrophic pathogens in transgenic carrot plants.     

Lipoxygenase Metabolites of α-linolenic Acid in the Development of Resistance in Pigeonpea, Cajanus cajan (L.) Millsp, Seedlings Against Fusarium udum Infection

Lipoxygenase (LOX) activity was measured in germinating pigeonpea Cajanus cajan seedlings, resistant (ICP-8863) and susceptible (ICP-2376) to wilt fungus, before and after infection with Fusarium udum. LOX activity was significantly higher in the resistant than in the susceptible cultivars of pigeonpea and was enhanced further in response to infection with Fusarium udum. This increase in LOX activity in the resistant cultivars of pigeonpea appears to be due to the induction of lipoxygenase isozymes in response to infection. Analysis of the endogenous LOX metabolites in pigeonpea seedlings revealed the predominant formation of 13-hydroperoxyoctadecadienoic acid (13-HPODE) in healthy seedlings and 13-hydroperoxyoctadecatrienoic acid (13-HPOTrE) in infected seedlings. Further studies on the effects of LOX metabolites on the growth and multiplication of Fusarium udum showed that HPOTrEs, LOX metabolites of -linolenic acid, are more anti-fungal compared to HPODEs, LOX metabolites of linoleic acid.     

Trichoderma sp. endochitinase and β-1,3-glucanase impede Rhizoctonia solani growth independently,and their combined use does not enhance impediment

Rhizoctonia solani causing rice sheath blight is responsible for significant crop yield losses. Trichoderma spp., biological control agents, have been reported to antagonize R. solani through coordinated action of several cell wall-degrading enzymes including endochitinase and β-1,3-glucanase. In this study two antifungal genes, encoding endochitinase and β-1,3-glucanase, isolated from Trichoderma sp. antagonistic to R. solani, were cloned individually in His-tagged expression vectors and mobilized in Escherichia coli for protein expression. The purified proteins assayed in vitro with R. solani impeded pathogen growth independently by causing hyphal distortions revealed through scanning electron microscopy. The combined use of endochitinase and β-1,3-glucanase did not enhance the inhibition. The distortions caused by endochitinase were due to catalytic activity of Glu172 and Asp241 residues on glycosidic linkages in chitin polymers, whereas Glu628, Tyr631, and Asp569 in β-1,3-glucanase targeted glucan polymers. The distinctions of this study from earlier reports are (a) chitin polymers in the R. solani cell wall are exposed and not embedded within the β-glucan matrix; (b) chitin and β-1,3-glucan are vital polymers in the R. solani cell wall, rather than chitin as the only main polymer; and (c) hyphal tips of R. solani remain unaffected after an antifungal assay with endochitinase and β-1,3-glucanase, instead of exhibiting distortion.     

Differential expression of a β-1,4-endoglucanase induced by diet change in the foliar nematode Aphelenchoides fragariae

We identified and characterized a β-1,4-endoglucanase, Afr-ENG-1, in the foliar nematode Aphelenchoides fragariae that is differentially expressed when the nematode feeds on fungi or plants. When individuals from hosta plants were transferred to a fungus culture, expression of the enzyme decreased 1,812-fold after five generations on the fungus diet. Afr-eng-1 was readily detected in the genome of 75% of nematodes from the plant population but only in 38% of the diet-changed population. The gene cannot be detected in nematodes maintained on fungus for over 100 generations. Diet was also associated with changes in nematode body size and in the severity of symptoms caused on hosta leaves. Plant-diet nematodes caused larger lesions and were longer and thinner than fungus-diet nematodes. Nematodes moved from a plant diet to a fungus diet for five generations had the same body size as the nematodes that had fed on the fungus for 100 generations. Full-length sequences of Afr-eng-1 were obtained and found to encode a glycosyl hydrolase family 5 protein. This is the first β-1,4-endoglucanase and plant-parasitism-related gene described in the genus Aphelenchoides.     

Activity of <Emphasis Type="Italic">ß</Emphasis>-1,3-glucanase and <Emphasis Type="Italic">ß</Emphasis>-1,4-glucanase in two potato cultivars following challenge by the fungal pathogen <Emphasis Type="Italic">Alternaria solani</Emphasis>

Early blight of potato, caused by Alternaria solani, is a ubiquitous disease in many countries around the world. Our previous screening of several Iranian potato cultivars found that variation in resistance exists between two cultivars: ‘Diamond’ and ‘Granula’. Cultivar Diamond is more resistant to multiple isolates of A. solani when compared to cv. Granula. Furthermore, we have found that different pathogen isolates have varying degrees of infection. We monitored the activities of two pathogen-related (PR) glucanase proteins in Diamond and Granula in response to two isolates of A. solani with different degrees of virulence. ß-1,3-glucanase and ß-1,4-glucanase activities were recorded in healthy and diseased leaves of potatoes up to 10 days after inoculation. Their activities were found to be higher in diseased leaves when compared to those of uninfected leaves. Our data suggest that significantly reduced activities of theses enzymes in potato could be related to a lower degree of resistance or an increased ability of a more aggressive isolate to suppress PR protein expression.     

Response mechanisms of nitrate and ammonia nitrogen concentrations to hydrological processes in the riparian hyporheic zone of pastoral areas北大核心CSCD

In pastoral and irrigated agricultural areas, nitrogen-containing livestock, poultry manure, and nitrogen fertilizers can enter the surface water and groundwater from the soil, and this is the main source of non- point source pollution in basins. The riparian hyporheic zone acts as an effective barrier to reduce the nitrogen pollution load. Understanding the mechanisms of the migration, transformation, and removal of nitrogen in riparian hyporheic zones is key to controlling nitrogen pollution in the whole basin. In this study, an upper reach of the Xilin River, located in typical pastoral areas, was selected and its water levels, ammonia (NH+4) and nitrate (NO-3) concentrations, as well as the related environmental factors of the river water and riparian groundwater during the summer flood season, were continuously monitored. Based on the high- solution measurements, a water flow and nitrogen reactive transport model of the riparian hyporheic zones was established using FEFLOW. The model fitted using the measured data was found to accurately reproduce the water level dynamics and two main nitrogen concentrations in the riparian hyporheic zone. The results indicate that there is a high risk of nitrogen pollution in the riparian zones during the summer flood season. The NH+4concentration in the riparian zones can increase from 0.2 mg · L − 1before rainfall events to 7.23 mg · L − 1after rainfall events, and the NO-3concentration can increase from 1 mg · L − 1to 8.27 mg · L − 1. Both measured and simulated results show that the nitrogen dynamics in the hyporheic zone are closely related to hydrological processes such as rainfall events and groundwater-surface water exchange. During rainfall events, NO-3with high mobility was found to infiltrate from the river and the ground surface into the riparian zone due to the leaching effect, resulting in a significant increase in the concentration. Meanwhile, the groundwater- river water exchange enhanced by rainfall events can further regulate NO-3and NH+4concentrations in the riparian hyporheic zone by controlling the input of nutrients and affecting the biogeochemical nitrogen cycles. This study preliminarily reveals the buffering mechanisms of pastoral riparian zones in the hydrological and biogeochemical processes involving nitrogen and provides scientific references for the nitrogen pollution control in pastoral areas. © 2023 Editorial office of Arid Zone Research. All rights reserved.     

Endo-1,3-β-glucanase and cellulase from Trichoderma harzianum: purification and partial characterization, induction of and biological activity against plant pathogenic Pythium spp.

There were indications that endo-1,3--glucanase (1,3-(1,3;1,4)--D-Glucan 3(4)-glucanohydrolase (EC 3.2.1.6)) and cellulase (1,4-(1,3;1,4)--D-Glucan 4-glucanohydrolase (EC 3.2.1.4)) activity of Trichoderma harzianum Rifai isolate T3 were induced in sphagnum peat moss cultivations and dual culture experiments by the presence of Pythium ultimum. Further, P. ultimum stimulated the germination of Trichoderma conidia. Endo-1,3--glucanase and cellulase were purified from T. harzianum isolate T3, known to control Pythium damping-off of cucumber seedlings. The enzymes were purified from the culture filtrate of the fungus by gel filtration and isoelectric focusing. The purified endo-1,3--glucanase was a small protein with a molecular mass of 17 kilodaltons and a pI of 5.0. Two cellulases were purified to homogeneity and had molecular masses of 40 and 45 kilodaltons respectively, and pI's of 6.4 and 7.6 respectively. Germination of encysted zoospores and elongation of germ tubes of a plant pathogenic Pythium isolate were inhibited by low concentrations of the purified enzymes. A strong synergistic effect was observed on the inhibition of cyst germination by a combination of the endo-1,3--glucanase and the fungicide Fongarid. Finally, a time-course study of colonization of the rhizosphere of cucumber seedlings showed that the active fungal mycelial biomass of a GUS-transformant of T. harzianum isolate T3 increased over four weeks. Trichoderma appeared to colonize healthy roots only superficially, whereas the mucilage of the root hairs and of distal parts of wounded areas or broken parts of the roots, were extensively colonized.     

Effect of the disodium salt of cyclopentene β,β′-triketone on the development of infections induced by TMV in the leaves of hypersensitive and susceptible tobacco plants

Journal of Plant Diseases and Protection - We show here that the disodium salt of 2-acetyl-5-chloro-4- hydroxycarbonylmethylthiocyclopent-4-ene-1,3-dione (salt 2) inhibits tobacco mosaic virus...     

Expression of the yeast Δ-9 desaturase gene in tomato enhances its resistance to powdery mildew

Tomato plants (Lycopersicon esculentumMill.) transformed with the yeast Δ-9 desaturase gene were found to display enhanced resistance to powdery mildew (Erysiphe polygoniDC. edmund. Salm.) and to possess higher levels of 16:1, 16:2 and 18:1 fatty acids. Lipid peroxides also increased. The improved resistance of the transgenic plants was apparently associated with the increases in 16:1 and 16:2 fatty acids.     

The response of phenolic compounds in grapes of the variety ‘Chardonnay’ (Vitis vinifera L.) to the infection by phytoplasma Bois noir

The study was carried out on phytoplasma susceptible grapevine variety ‘Chardonnay’ (Vitis vinifera L.). The changes in total and individual phenolics, with a focus on hydroxycinnamic acids, flavanols and flavonol contents, were studied in phytoplasma-symptomatic and non-symptomatic berries of Bois noir (BN) infected and uninfected vines. Evident responses to BN infection at veraison have been monitored in a decreased accumulation of caftaric and coutaric acids, p-coumaroyl hexose, procyanidin B1, procyanidin trimer as well as of quercetin-3-O-glucoside, quercetin-3-O-glucuronide and quercetin-3-O-xyloside. At berry softening BN infection statistically increased the content of total phenolics, hydroxycinnamic acids and flavanols, but decreased the flavonol contents, especially at phytoplasma-symptomatic berry skins. Later, at harvest, the BN infection caused an additional significant decrease of coutaric acid and p-coumaroyl pentose contents, moreover of procyanidin B1 and procyanidin dimmers (1, 2, and 3), trimer, kaempferol-3-O-glucoside and of most identified quercetins, except of quercetin-3-O-xyloside. At harvest, non-symptomatic berries from infected plants showed similar dynamics in the total phenolic content compared to berry skins from uninfected plants, but in total flavanols and flavonols content similarity to those symptomatic was observed. The latter decreases grape quality and its antioxidant potential. The Bois noir disease showed specific, local and growth-phase-induced responses regarding the content of phenolics in berry skins, where in particular the differences between phytoplasma-symptomatic and non-symptomatic grapes have to be underlined.     

Response to the letter on “Climatic distribution of citrus black spot caused by <Emphasis Type="Italic">Phyllosticta citricarpa.</Emphasis> A historical analysis of disease spread in South Africa” by Fourie et al. (2017)

In a previous study, Martínez-Minaya et al. (European Journal of Plant Pathology 143, 69–83, 2015) performed an analysis of climate-based distribution of citrus black spot (CBS) in South Africa. It was found that CBS was initially confined to humid areas with summer rainfall, but later spread to arid steppe and even desert climates. A strong spatial autocorrelation of CBS distribution was found. Fourie et al. (European Journal of Plant Pathology, 2017) take a critical view of our study, but without presenting any analysis of results to refute our findings. Furthermore, Fourie et al. (European Journal of Plant Pathology, 2017) appear to have misunderstood our work, since many of their criticisms relate to the potential distribution of CBS in Europe, which is beyond the scope of our original study. Fourie et al. (European Journal of Plant Pathology, 2017) highlight the limitations of climate classifications in species distribution modelling. However, this was made explicit in our study, indicating that it was a preparatory work and further advanced modelling studies, including spatial effects, will be needed. Fourie et al. (European Journal of Plant Pathology, 2017) incorrectly assume that we used all of South Africa as the background in the spatial autocorrelation analysis. However, only citrus areas were used and a strong spatial autocorrelation was detected at all distances evaluated. Contrary to what Fourie et al. (European Journal of Plant Pathology, 2017) suggest, similar climate distributions of CBS were obtained at 5′ and 30′ resolution, and also with the national land-cover map of South Africa. The figure comparison presented by Fourie et al. (European Journal of Plant Pathology, 2017) appears to ignore the fact that the maps we used were grid cells of 10 × 10 km and not the line polygons they suggest. Therefore, we consider the conclusions from the Martínez-Minaya et al. (European Journal of Plant Pathology 143, 69–83, 2015) remain entirely valid.