Molecular cloning,recombinant gene expression,and antifungal activity of cystatin from taro (<Emphasis Type="Italic">Colocasia esculenta</Emphasis> cv. Kaosiung no. 1)

A cDNA clone, designated CeCPI, encoding a novel phytocystatin was isolated from taro corms (Colocasia esculenta) using both degenerated primers/RT-PCR amplification and 5-/3-RACE extension. The full-length cDNA gene is 1,008 bp in size, encodes 206 amino acid residues, with a deduced molecular weight of 29 kDa. It contains a conserved reactive site motif Gln-Val-Val-Ser-Gly of cysteine protease inhibitors, and another consensus ARFAV sequence for phytocystatin. Sequence analysis revealed that CeCPI is phylogenetically closely related to Eudicots rather than to Monocots, despite taro belonging to Monocot. Recombinant GST–CeCPI fusion protein was overexpressed in Escherichia coli and its inhibitory activity against papain was identified on gelatin/SDS-PAGE. These results confirmed that recombinant CeCPI protein exhibited strong cysteine protease inhibitory activity. Investigation of its antifungal activity clearly revealed a toxic effect on the mycelium growth of phytopathogenic fungi, such as Sclerotium rolfsii Sacc. etc., at a concentration of 80 g recombinant CeCPI/ ml. Moreover, mycelium growth was completely inhibited and the sclerotia lysed at a concentration of 150–200 g/ml. Further studies have demonstrated that recombinant CeCPI is capable of acting against the endogenous cysteine proteinase in the fungal mycelium.     

Oxidative Stress in Relation to Lipid Peroxidation, Sclerotial Development and Melanin Production by Sclerotium rolfsii

Melanin pigments constituted 13.9% of the chemical composition of the sclerotial walls of Sclerotium rolfsii and was associated with proteins, reducing sugars and amino acids. The lipid and ash contents in the sclerotial walls were double those in the hyphal walls of the fungus. Increasing age of the culture and maturation of the sclerotia were always accompanied by elevation of lipid peroxides and melanin pigments. Such behaviour may indicate that lipid peroxidation and melanin formation are operating in parallel during sclerotial biogenesis and maturation. These two processes depend on the theory of oxidative stress, as affected by growth conditions. Both processes could be stopped or sharply retarded when subjected to some antioxidant growth factors such as vitamins (ascorbic acid), micro-elements (selenium) and sulfhydryl compounds (glutathione). A clear relation between oxidative stress, myceliogenic germination and lytic activity via melanin production was observed. This finding appears promising in applying a new control measure against diseases caused by sclerotia-producing fungi without using traditional toxic fungicides.     

Bahiagrass,Corn, Cotton Rotations,and Pesticides for Managing Nematodes,Diseases, and Insects on Peanut

Florunner peanut was grown after 1 and 2 years of Tifton 9 bahiagrass, corn, cotton, and continuous peanut as whole-plots. Pesticide treatments aldicarb (3.4 kg a.i./ha), flutolanil (1.7 kg a.i./ha), aldicarb + flutolanil, and untreated (control) were sub-plots. Numbers of Meloidogyne arenaria second-stage juveniles in the soil and root-gall indices of peanut at harvest were consistently lower in plots treated with aldicarb and aldicarb + flutolanil than in flutolanil-treated and untreated plots. Percentages of peanut leaflets damaged by thrips and leafhoppers were consistently greater in flutolaniltreated and untreated plots than in plots treated with aldicarb or aldicarb + flutolanil but not affected by cropping sequences. Incidence of southern stem rot was moderate to high for all chemical treatments except those that included flutolanil. Stem rot loci were low in peanut following 2 years of bahiagrass, intermediate following 2 years of corn or cotton, and highest in continuous peanut. Rhizoctonia limb rot was more severe in the peanut monoculture than in peanut following 2 years of bahiagrass, corn, or cotton. Flutolanil alone or combined with aldicarb suppressed limb rot compared with aldicarb-treated and untreated plots. Peanut pod yields were 4,186 kg/ha from aldicarb + flutolanil-treated plots, 3,627 kg/ha from aldicarb-treated plots, 3,426 kg/ha from flutolanil-treated plots, and 3,056 kg/ha from untreated plots. Yields of peanut following 2 years of bahiagrass, corn, and cotton were 29% to 33% higher than yield of monocultured peanut.     

植物病原真菌的菌核研究进展

真菌菌核是相关真菌在特殊环境下由营养体菌丝交织和聚集形成的具有抵御恶劣环境能力的休眠结构,在真菌生活史及病原真菌的病害循环中具有重要的生物学和生态学意义。许多引起严重植物病害的病原真菌能够形成菌核,这类真菌通过菌核度过逆境。菌核在适宜条件下萌发形成子囊盘和孢子或菌丝,造成植物的侵染。本文从形成菌核的真菌种类、生物学特性、黑色素及分子生物学等方面进行综述,旨在为深入研究真菌菌核及其在真菌学、植物病理学及药用真菌学等领域应用提供研究思路和参考依据。     

Evidence of Phytoalexins in Rhizome of Atractylodis Maceocephalae Koidz Infected with Sclerotium rolfsii sacc Following Treatment with the Polysaccharides of Chrysanthemum indicum

Atractylenolides compounds (atractylenolide‐I, atractylenolide‐II and atractylenolide‐III) extracted from Atractylenolides macrocephala were analysed for their differential presence in the rhizome of both the infected and healthy plants and its fungitoxicity on prophylactic treatment with polysaccharides of Chrysanthemum indicum against Sclerotium rolfsii. It was evident that the plant under stress by pathogen has instigated the significant synthesis and accumulation of atractylenolide‐II and atractylenolide‐III in the rhizome of plants as a result of elicitation with polysaccharides of C. indicum. Our results also showed that the absence of atractylenolide‐II in the rhizome made the plant susceptible to S. rolfsii. Furthermore, on in vitro antifungal studies, exogenous application of atractylenolide‐II with the concentration of 200 μg/ml showed a significant inhibitory effect on mycelial growth by achieving a 77.23% antifungal activity rate, and a minimal inhibitory effect at a concentration of 12.5 μg/ml with atractylenolide‐II. To our knowledge, this novel study on phytoalexins in A. macrocephala suggested that A. macrocephala plants produce elevated levels of atractylenolide‐II and atractylenolide‐III in a pattern typical of phytoalexins, in response to an eliciting treatment after infection.     

Biochemical Investigations of Sclerotial Exudates of Sclerotium rolfsii and their Antifungal Activity

Exudates from sclerotia of two Sclerotium rolfsii isolates (one causing collar rot in Cicer arietinum, isolate VC971, and the other leaf spots in Rauvolfia serpentina, isolate VL016) were assayed for their antifungal activity against 26 fungi consisting of plant parasites as well as saprophytes. Spore germination of all the test fungi was affected by the exudates reaching 100% in some cases. Foliar spray with exudates of isolate VL016 significantly reduced disease incidence of balsam (Impatiens balsamina) powdery mildew caused by Erysiphe cichoracearum and pea (Pisum sativum) powdery mildew caused by Erysiphe pisi, under field conditions. Characterization of exudates from 25 isolates of S. rolfsii revealed pH ranging from 3.8 to 5.3 and colour from light yellow to deep yellow. Among the phenolic acids found in the exudates were tannic, gallic, caffeic, vanillic, ferulic, chlorogenic and cinnamic acids. Oxalic acid was also found in varied amounts. Among the phenolic acids, ferulic acid was found to be present at high concentration in exudates of most isolates (3.9–153.4 μg/ml). The antioxidant properties of phenolics, which generally inhibit fungal morphogenesis including spore germination along with the antifungal nature of some phenolics are chiefly attributed to the inhibitory effect of sclerotial exudates of S. rolfsii. Additionally, both the isolates VC971 and VL016 showed almost similar antifungal activities despite they are of different origin and thereby demonstrate the antifungal nature of sclerotial exudates.     

Molecular insights into development of Trichoderma interfusants for multistress tolerance enhancing antagonism against Sclerotium rolfsii Sacc

The current study aimed at developing diverse Trichoderma fusants for fungicides, drought, and salt tolerance with enhanced antagonistic activity against Sclerotium rolfsii Sacc. Trichoderma virens NBAII Tvs12 (mycoparasitic) and Trichoderma koningii MTCC796 (multistress tolerant) were used as parental strains for development of interspecific protoplast fusants. A total of 36 stable fusants were used for mycoparasitism, fungicides, and abiotic stresses (drought and salt) tolerance. The results revealed 20 homozygous progenies showing characteristics of either one parental strain and 14 heterozygous mutants depicting traits of both parental strains. A novel concept of inhibition coefficient was established using growth-related key parameters that represent the pathogen biology and the biocontrol-related biophysics of Trichoderma fusants. The results indicated a differential inhibition coefficient of the test pathogen and the highest (92.88%) inhibition coefficient of S. rolfsii was observed by interstable fusant Fu21. It also grew better under fungicides and abiotic stress (drought and salt) conditions. The molecular characterization and heterozygosity analysis evidenced the highest observed heterozygosity (0.5441) and gene flow (0.3872) in stable heterozygous Fu21. Principal coordinates analysis exhibited 62.7% of total variability. The ecofriendly heterozygous Trichoderma fusant (Fu21) might be useful for biocontrol of stem rot disease under adverse conditions or as a part of integrated disease management.     

Comparative studies on simple lipids ofSclerotium cepivorum,the causal agent of white rot of onion,and other fungi of the class Deuteromycetes

Summary The simple lipids ofSclerotium cepivorum, the causal agent of white rot of onion and nine other fungal species of the same class were investigated. The fatty acid composition of the simple lipids of these fungi were determined by GLC. The main fatty acids common to these fungal species were C₁₆ (saturated) and C₁₈ (unsaturated) acids. The sterol fraction was isolated by column chromatography and its components were detected by GLC and mass spectrometry. Ergosterol and γ-Ergostenol were found mostly in all fungal species under investigation. However, two fungal species namelyAlternaria alternata andScolecobasidium constrictum showed no Ergosterol.