Mycoparasitism of Endophytic Fungi Isolated From Reed on Soilborne Phytopathogenic Fungi and Production of Cell Wall-Degrading Enzymes In Vitro

Antagonism of three endophytic fungi isolated from common reed (Phragmites australis) against eight soilborne pathogenic fungi was investigated on potato dextrose agar by light microscopy, scanning electron microscopy, and transmission electron microscopy. Inhibitory zones were not observed. The microscopical studies suggested that the endophytes inhibit growth of soilborne pathogens by means of coiling around hyphae and, after penetration, the degradation of hyphal cytoplasm. Since penetration of hyphae seems to play a major role in parasitism, we studied the production of cell wall degrading enzymes by the three endophytes. Choiromyces aboriginum produced higher activities of β-1,3-glucanases compared to Stachybotrys elegans and Cylindrocarpon sp. For C. aboriginum and S. elegans, colloidal chitin was the best substrate for the induction of β-1,3-glucanases and chitinases, respectively. This result suggests that mycoparasitism by endophytes on soilborne plant pathogens can be explained by their mycoparasitic activity.     

Purification and Characterization of Extracellular Pectinolytic Enzymes Produced by Sclerotinia sclerotiorum

An exopolygalacturonase (exoPG) and an exopolymethylgalacturonase (exoPMG) produced by Sclerotinia sclerotiorum have been purified by ammonium sulfate precipitation, gel filtration, and ion exchange chromatography. The exoPG and the exoPMG were purified 66- and 50-fold, respectively, by using a series of separation procedures that included ammonium sulfate precipitation and gel chromatography. Molecular masses of the native proteins were 68 kDa for exoPG and 140 kDa for exoPMG. The pH optima of the enzymes were about pH 5, and their optimum temperature was 45°C. Activities of both enzymes were inhibited by Hg²⁺, Zn²⁺, Cu²⁺, and p-chloromercuribenzoate. ExoPMG activity, in contrast to exoPG activity, was stimulated by Mn²⁺ and Co²⁺. ExoPMG hydrolyzed only citrus pectin, while exoPG degraded sodium polygalacturonate and, to a lesser extent, citrus pectin. The exo mode of action of the enzymes was revealed by thin-layer chromatography of substrate hydrolysates. Antibodies raised against each purified protein exhibited no cross-reaction, thus confirming the biochemical specificities of the enzymes.     

Yeast Spheroplasts Formed by Cell Wall-Degrading Enzymes from Oerskovia sp

Oerskovia sp. produces inducible extracellular enzymes which degrade the walls of various yeasts. Yeast spheroplasts are formed from both log- and stationary-phase cells.     

Regulation by Galacturonic Acid of Pectinolytic Enzyme Production by Sclerotinia sclerotiorum

Production of polygalacturonases and pectinases from Sclerotinia sclerotiorum was induced in vitro by galacturonic acid. The inductive effect of galacturonic acid was abolished by the presence of glucose, leading to a basal enzyme production. Zymograms of extracellular enzymes showed that galacturonic acid induced the synthesis of six polygalacturonase and one pectin-methylesterase isoforms. Immunoblotting revealed that an exo-polygalacturonase and an exo-polymethylgalacturonase were secreted in all conditions. They are not glucose repressed and not regulated by galacturonic acid. These constitutive enzymes provide the pathogen with the inherent ability to release galacturonic acid from plant cell walls and to trigger inducible enzyme synthesis. Received: 1 December 1995 / Accepted: 3 January 1996     

Secretion of pectic isoenzymes by Sclerotinia sclerotiorum

Abstract Cultures of Sclerotinia sclerotiorum grown on different pectin-related polysaccharides (citrus pectin, apple pectin, sodium polygalacturonate), carboxymethylcellulose (CMC) or glucose as the only carbon source were examined daily for polygalacturonase and pectinase activities. Electrophoretic forms of polygalacturonase and pectin methylesterase activities were revealed using analytical IEF and sodium polygalacturonate and citrus pectin as substrates in overlay gels. A sequence in the production of pectic enzymes and isoenzyme synthesis was found in pectic-polymer cultures corresponding to the induction of several isoenzymes. Enzyme activities in glucose media were associated with three polygalacturonase and two pectinmethylesterase isoforms which were produced constitutively. Sodium-dodecyl-sulphate polyacrylamide-gel electrophoresis followed by immunoblotting with polyclonal antibodies against an exo-polymethylgalacturonase and an exo-polygalacturonase revealed that these exo-enzymes were secreted from the beginning of cultivation in the different culture media showing characteristics of constitutive enzymes.     

Electrophoretic Karyotypes of Sclerotinia sclerotiorum

Electrophoretic karyotypes (EKs) of 83 isolates were variable within agricultural and natural populations of Sclerotinia sclerotiorum, as well as among S. sclerotiorum, Sclerotinia minor, and Sclerotinia trifoliorum. Variation in EKs was not observed within six mitotic or three meiotic lineages of isolates. EKs of 8 to 10 chromosome-sized DNAs were observed. Homologous and heterologous probes hybridized to four linkage groups.     

Suppression by Zygorrhynchus moelleri of Growth and Sclerotium Production by Rhizoctonia solani and Sclerotinia sclerotiorum

As indicated by reduced cellulolysis, Zygorrhynchus moelleri suppressed mycelial growth in Rhizoctonia solani and Sclerotinia sclerotiorum. Sclerotium production by both pathogenic fungi was also reduced by Z. moelleri in dual sand-oatmeal cultures. The viability of sclerotia produced by S. sclerotiorum, but not those produced by R. solani, was greatly reduced. Sclerotium production by S. sclerotiorum on celery and tomato segments was reduced to a much greater extent when Z. moelleri was applied to the plant tissue 24 h before the pathogen than when applied at the same time or 24 h after the pathogen.     

Agrobacterium-mediated transformation of Sclerotinia sclerotiorum

Ascospores from the phytopathogenic fungus Sclerotinia sclerotiorum were transformed to hygromycin B resistance by co-cultivation with Agrobacterium tumefaciens. Transformed spores germinated and grew on PDA supplemented with 100 ug/ml hygromycin B. The presence of mitotically stable hph gene integration at random sites in the genome was confirmed by PCR and Southern blot analysis. A transformation frequency of 8 x 10(-5) was achieved in five separate experiments. This study is the first report of success co-cultivating A. tumefaciens with S. sclerotiorum. This report of a reproducible Agrobacterium-mediated transformation method should allow the development of T-DNA tagging as a system for insertional mutagenesis in S. sclerotiorum and provide a simple and reliable method for genetic manipulation.     

Purification of the beta-glucosidase from Sclerotinia sclerotiorum

A beta-glucosidase (EC 3.2.1.21) has been isolated from culture filtrates of the fungus Sclerotinia sclerotiorum. The protein was purified by gel filtration on a column of Bio-Gel P-300 and by ion exchange chromatography on DEAE-Bio-Gel A. The molecular weight, determined by gel filtration, was 240,000. Km values for the enzyme towards p-nitrophenyl-beta-D-glucoside and cellobiose were respectively 0.10 mM and 1.23 mM. The beta-glucosidase activity was found to be strongly associated with a beta-xylosidase (EC 3.2.1.37) activity, suggesting that both activities could be represented in a single protein complex.     

Salicylate Degradation by the Fungal Plant Pathogen Sclerotinia sclerotiorum

The fungal plant pathogen Sclerotinia sclerotiorum was studied to determine its ability to degrade salicylate, an important defense-signaling molecule in plants. S. sclerotiorum D-E7 was grown at 25 °C in an undefined medium (50 ml) containing minerals, 0.1 % soytone, 50 mM MES buffer (pH 6.5), 25 mM glucose, and 1 mM salicylate. Glucose, oxalate, and salicylate concentrations were monitored by HPLC. S. sclerotiorum D-E7 was found to be active in salicylate degradation. However, salicylate alone was not growth supportive and, at higher levels (10 mM), inhibited glucose-dependent growth. Biomass formation (130 mg [dry wt] of mycelium per 50 ml of undefined medium), oxalate concentrations (~10 mM), and culture acidification (final culture pH approximated 5) were essentially the same in cultures grown with or without salicylate (1 mM). Time-course analyses revealed that salicylate degradation and glucose consumption were complete after 7 days of incubation and was concomitant with growth. Trace amounts of catechol, a known intermediate of salicylate metabolism, were detected during salicylate degradation. Overall, these results indicated that S. sclerotiorum has the ability to degrade salicylate and that the presence of low levels of salicylate did not affect growth or oxalate production by S. sclerotiorum.     

粉红黏帚霉67-1菌株寄生核盘菌菌核的酶学动态研究

对粉红黏帚霉67-1菌株侵染核盘菌菌核过程的多种细胞壁降解酶活性进行了连续测定,以研究几丁质酶等在这一寄生互作体系中的可能作用。结果表明:葡聚糖酶活性变化表现活跃,且随寄生过程呈增加趋势,配对法T检验结果表明,第10d的处理与对照酶活性差异达到最大;几丁质酶、蛋白酶活性变化表现较低,而纤维素酶未检测得到。酶学动态变化与之前石蜡切片显微观察的结果在时间上表现一致;认为葡聚糖酶可能是粉红黏帚霉67-1菌株寄生核盘菌菌核的关键酶。     

Cell Wall-Degrading Enzymes Enlarge the Pore Size of Intervessel Pit Membranes in Healthy and Xylella fastidiosa-Infected Grapevines

The pit membrane (PM) is a primary cell wall barrier that separates adjacent xylem water conduits, limiting the spread of xylem-localized pathogens and air embolisms from one conduit to the next. This paper provides a characterization of the size of the pores in the PMs of grapevine (Vitis vinifera). The PM porosity (PMP) of stems infected with the bacterium Xylella fastidiosa was compared with the PMP of healthy stems. Stems were infused with pressurized water and flow rates were determined; gold particles of known size were introduced with the water to assist in determining the size of PM pores. The effect of introducing trans-1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid (CDTA), oligogalacturonides, and polygalacturonic acid into stems on water flux via the xylem was also measured. The possibility that cell wall-degrading enzymes could alter the pore sizes, thus facilitating the ability of X. fastidiosa to cross the PMs, was tested. Two cell wall-degrading enzymes likely to be produced by X. fastidiosa (polygalactuoronase and endo-1,4- β -glucanase) were infused into stems, and particle passage tests were performed to check for changes in PMP. Scanning electron microscopy of control and enzyme-infused stem segments revealed that the combination of enzymes opened holes in PMs, probably explaining enzyme impacts on PMP and how a small X. fastidiosa population, introduced into grapevines by insect vectors, can multiply and spread throughout the vine and cause Pierce''s disease.In grapevine (Vitis vinifera) stems, water moves with the transpiration stream from one vessel to the next, traversing the scalariform bordered pits in the secondary wall (Mullins et al., 1992; Stevenson et al., 2004b). Water must also pass through the pit membrane (PM) that occurs between a pit pair (i.e. opposing pits in adjacent vessels; Mauseth, 1988; Dickison, 2000). A PM is the shared primary cell walls and middle lamella of vessels that were not covered with the secondary wall of neighboring vessel elements as the development of the water conducting system progressed. PMs are composed of cellulose microfibrils embedded in a polysaccharide matrix of hemicellulose and pectins (Mauseth, 1988; Fisher, 2000; Tyree and Zimmermann, 2002). The fine mesh-like polysaccharide structure of PMs provides minute openings (pores) through which water can move with minimal restriction to other vessels or neighboring parenchyma cells. In angiosperm trees, the diameters of most of these pores have been described to vary between 5 and 20 nm (Choat et al., 2003, 2004), but pores of up to several hundred nanometers have occasionally been observed (Sperry et al., 1991; Sano, 2005; Wheeler et al., 2005). The small size of the PM pores is a safety mechanism that limits the expansion of gas bubbles from one cavitated vessel to its neighbors and the movement of pathogens from one infected, water-filled vessel to its neighbors as water moves through the xylem system (Sperry and Tyree, 1988; Nakaho et al., 2000; Tyree and Zimmermann, 2002). It is thought that pore size is largely controlled by the physical arrangement of hydrated pectins and the cross-links that they establish with themselves and other polysaccharides in the PM (Fleischer et al., 1999; Zwieniecki et al., 2001). The infiltration of vessels with solutions having modifications in ion content or pH causes changes in hydraulic resistance that are consistent with swelling or shrinking of pectins and the consequent changes in PM pore size (Zwieniecki et al., 2001).The xylem-limited bacterium Xylella fastidiosa is the causal agent of Pierce''s disease (PD) of grapevines. X. fastidiosa is vectored by sharpshooter (Cicadellidae) and spittlebug (Cercopidae) insects that feed on xylem sap and introduce the bacteria into xylem vessels (Varela et al., 2001). In order for the bacterial population to become systemic, individual bacterial cells must cross the PMs that separate two adjoining vessels. X. fastidiosa is a rod-shaped bacterium with dimensions ranging from 250 to 500 × 1,000 to 4,000 nm (Mollenhauer and Hopkins, 1974), making them too large to pass freely through the majority of the PM pores that have been described in angiosperms. Nevertheless, evidence of X. fastidiosa cells traversing PMs and gaining access to an adjacent vessel has been reported (Newman et al., 2003). This intervessel movement of X. fastidiosa cells was observed too frequently by Newman et al. (2003) to be considered the result of random encounters with damaged PMs; thus, they proposed that X. fastidiosa is able to degrade the grapevine PM. The involvement of cell wall-degrading enzymes during PD first had been proposed based on indirect evidence from the development of internal symptoms and the location of bacteria in X. fastidiosa-infected shoots, but the absence of evidence for bacterial enzyme production limited wide acceptance of this idea (Hopkins, 1989; Fry and Milholland, 1990a; Purcell and Hopkins, 1996). However, the observation of intervessel X. fastidiosa movement described above and reports that the X. fastidiosa genome contains several genes similar to those encoding cell wall-degrading polygalacturonase (PG) and endo-1,4- β -glucanase (EGase) in other bacteria (Simpson et al., 2000; Wulff et al., 2003) suggested the contrary. Indeed, a X. fastidiosa mutant disrupted in the pglA gene, which encodes an endo-PG, was restricted to the point of inoculation and unable to move systemically in grapevine, indicating that PG plays a major role in vessel-to-vessel movement (Roper et al., 2007). Furthermore, recombinant X. fastidiosa PG (Roper et al., 2007) and EGase (this study) are capable of digesting polygalacturonic acid (PGA) and β -1,4 linked glucans, respectively. The recent detection of PG in the xylem sap of infected vines and less severe symptom development in transgenic grapevines expressing a pear (Pyrus communis) PG-inhibiting protein (pear PGIP [pPGIP]) also suggests that X. fastidiosa uses cell wall-degrading enzymes to open up PM pores to facilitate vessel-to-vessel movement (Agüero et al., 2005).We have reported that during early stages of X. fastidiosa infection, some stems presented exceptionally high hydraulic conductivities (higher than comparable healthy stems), which was attributed to enzymatic digestion of the PMs (Perez-Donoso et al., 2007). In this study, the size of PM pores in healthy and X. fastidiosa-infected stems was approximated by flushing the xylem of grapevine stem explants from the base with water containing particles of known size and determining if the particles could be recovered in the water collected from the distal ends of the explants. We also tested the ability of the PG and EGase activities that have been reported in X. fastidiosa (Roper, 2006; Roper et al., 2007) for digesting the intervessel PM and increasing the size of the PM pores and report that the pPGIP inhibits X. fastidiosa’s PG, presumably explaining why expression of the pPGIP-encoding gene in transgenic grapevines suppresses PD development (Agüero et al., 2005). Finally, the effects of stem infiltration with the chelator trans-1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid (CDTA), PGA (a model homogalacturonan pectin), or oligogalacturonides (OGAs; potential signal molecules generated when PGA is digested by PG) on PM pore size were also evaluated.     

菌核病防治研究进展

菌核病是一种寄主种类广泛的重大植物病害,可侵染450多种重要作物和草类,在我国每年给油菜、大豆以及多种蔬菜带来10～30亿元的损失.介绍了菌核病的症状、危害以及致病机理等,概述了主要的防治措施,并报道了国内外在关于菌核病生物防治、转基因育种、分子机理等方面的研究进展.     

The 203 kbp Mitochondrial Genome of the Phytopathogenic Fungus Sclerotinia borealis Reveals Multiple Invasions of Introns and Genomic Duplications

Here we report the complete sequence of the mitochondrial (mt) genome of the necrotrophic phytopathogenic fungus Sclerotinia borealis, a member of the order Helotiales of Ascomycetes. The 203,051 bp long mtDNA of S. borealis represents one of the largest sequenced fungal mt genomes. The large size is mostly determined by the presence of mobile genetic elements, which include 61 introns. Introns contain a total of 125,394 bp, are scattered throughout the genome, and are found in 12 protein-coding genes and in the ribosomal RNA genes. Most introns contain complete or truncated ORFs that are related to homing endonucleases of the LAGLIDADG and GIY-YIG families. Integrations of mobile elements are also evidenced by the presence of two regions similar to fragments of inverton-like plasmids. Although duplications of some short genome regions, resulting in the appearance of truncated extra copies of genes, did occur, we found no evidences of extensive accumulation of repeat sequences accounting for mitochondrial genome size expansion in some other fungi. Comparisons of mtDNA of S. borealis with other members of the order Helotiales reveal considerable gene order conservation and a dynamic pattern of intron acquisition and loss during evolution. Our data are consistent with the hypothesis that horizontal DNA transfer has played a significant role in the evolution and size expansion of the S. borealis mt genome.     

Changes in Cell Membrane Permeability in Sunflower Hypocotyls Infected with Sclerotinia sclerotiorum

Influx and efflux of water and urea and electrolyte leakage are less for sunflower (Helianthus annuus) hypocotyl sections above lesions caused by Sclerotinia sclerotiorum than for those from healthy plants. Urea uptake by sections above lesions is reduced (celery, squash, and tomato) or unchanged (bean) in other hosts after Sclerotinia infection. Efflux of urea from sunflower hypocotyls is biphasic, suggesting diffusion in series from two cellular compartments (cytoplasm and vacuole). Efflux during the fast phase was 7 to 20 times greater than that during the slow phase. No difference was noted in urea efflux from healthy and diseased tissues during the slow phase. However, efflux during the fast phase from diseased tissues was slower than from healthy tissues, suggesting that the increased resistance to diffusion of urea in host cells above lesions resides in the plasmalemma. Water movement across cell membranes of healthy and diseased sunflower hypocotyls was reduced when tissues were treated with p-hydroxymercuribenzoate.     

黄绿木霉菌代谢产物对大豆菌核病核盘菌的抑菌能力研究

利用室内实验的方法,研究了黄绿木霉对大豆菌核病菌的抑菌能力。黄绿木霉在与核盘菌（Sclerotinia sderotiorum）对峙培养过程中,5-7d即可将菌核病菌完全覆盖,在形成的菌核上会有黄绿木霉孢子出现,扫描电镜中观察到核盘菌的菌丝体发生变形;其代谢产物在经121℃处理25min后,抑菌作用仍可高达100％;在对黄绿木霉发酵过程中,利用PD培养基即可达到有效的控制大豆菌核病菌菌丝生长的目的,而且利用PD培养基发酵3-4d的黄绿木霉代谢产物抑菌效果最好,抑菌中浓度最低,发酵时间过长与过短,均不利于对病原菌的抑制。经黄绿木霉代谢产物处理的菌核菌电导率与呼吸强度均发生变化,在光学显微镜下观察到菌核菌菌体发生变化,且处理后的菌核菌致病力明显降低。     

Phenolic Compounds in Stems of Sunflower Plants Inoculated with Sclerotinia sclerotiorum and their Inhibitory Effects on the Fungus

Sunflower plants at I (vegetative), II (closed flower-buds) and III (open flower-buds) growth stages were experimentally inoculated with Sclerotinia sclerotiorum. At the 5th day after inoculation, all plants from I, III and 70 % of II (II_T) did not present wilt symptoms while 30 % of age II plants (II_s) presented severe wilt symptoms in all their leaves and had collapsed. Total soluble phenols were measured in the “lesion” (L) and its “healthy” (H) surrounding zones of all inoculated and control plants. A significant increase in phenols was found in the L and H zones of tolerant plants (I, II_t and III). The corresponding methanolic extracts showed a strong inhibitory effect on the mycelial growth (MG) of the fungus. On the other hand no statistically significant increase took place in the L zones from susceptible plants; their methanolic extracts did not inhibit MG. The chemical analysis of methanolic extracts showed that the isochlorogenic acid was the more inhibitory compound. It significantly accumulated in the L of tolerant plants but did not in those of susceptible ones.     

Factors Affecting Biological Control of Sclerotinia sclerotiorum by Fungal Antagonists

Studies were conducted to determine the effects of soil moisture (9, 16 or 24% w/w) and temperature (5, 15, 20 or 25°C) on the control of sclerotia of Sclerotinia sclerotiorum by five fungal agents in sterile and natural field soil. All five biocontrol agents were effective in reducing the survival of sclerotia of S. sclerotiorum in sterile soil under dry (9% moisture) or wet (24% moisture) conditions at 20°C, but only Coniothyrium minitans was effective in natural soil. Coniothyrium minitans was the most effective in reducing sclerotial viability at the temperature range of 15–25°C. Trichoderma virens was effective against sclerotia of S. sclerotiorum to a lesser extent than C. minitans , and in non-autoclaved soil, it performed best at 25°C. Although Epicoccum purpurascens , Talaromyces flavus and Trichothecium roseum were effective against sclerotia of S. sclerotiorum in some instances, they were less effective than C. minitans and T. virens . Sclerotia of S. sclerotiorum conditioned for myceliogenic germination were more vulnerable to attack by the biocontrol agents than dormant sclerotia. The implications are discussed with respect to enhancement of biological control of crop diseases caused by S. sclerotiorum in different geographic regions.