Induced Resistance as a Possible Means to Control Diseases of Strawberry Caused by Phytophthora spp.

Two putative elicitors of disease resistance (acibenzolar-S-methyl and chitosan) were tested for their effect on crown rot (Phytophthora cactorum) in strawberry. The effect of both compounds was enhanced when the time between treatment and inoculation was prolonged from 2 to 20 days. There were no significant differences between treatments when the concentration of acibenzolar-S-methyl was increased from 10 to 1,000 µg a.i./plant. The lowest tested concentrations of chitosan (10 and 50 µg a.i./plant) resulted in a lower disease score compared with the highest concentrations (250 or 1,000 µg a.i./plant). There were no differences in disease score between treatment with fosetyl-Al, acibenzolar-S-methyl, or chitosan when applied 5 or 15 days before inoculation. The effect of acibenzolar-S-methyl and chitosan also was tested against P. fragariae var. fragariae in alpine strawberry (Fragaria vesca var. alpina cv. Alexandria). Chitosan had no effect, whereas fosetyl-Al and all treatments with acibenzolar-S-methyl (50 or 250 µg a.i./plant; 5, 10, 20, or 40 days before inoculation) reduced the severity of the disease. There were no significant differences between acibenzolar-S-methyl and fosetyl-Al when applied at the same time. Acibenzolar-S-methyl and chitosan at concentrations of 0.5, 5, 50, and 500 µg a.i. ml-1 in V8 juice agar were tested for possible effects on P. cactorum and P. fragariae var. fragariae in vitro. Only chitosan at concentrations of 50 and 500 µg a.i. ml-1 had a growth-retarding effect on P. cactorum. Both acibenzolar-S-methyl and chitosan at a concentration of 500 µg a.i. ml-1 reduced the growth rate of P. fragariae var. fragariae.

Chilling resistances of isolates of Pythium ultimum var. ultimum from the Arctic and Temperate Zones.

Chilling resistances in moss pathogenic fungi, Pythium ultimum var. ultimum, from Longyearbyen, Svalbard (78 degree N, 15 degree E), located in the Arctic Zone and in the same isolates from Temperate Zone, were determined. Both strains had similar optimum growth temperatures. However, the strains from Svalbard could grow and survive at 0 - 5 degrees C. In addition, chilling treatment induced irregular mycelial morphology in the Arctic isolates. On the other hand, the isolates from Japan did not grow at temperatures below 5C and were destroyed after chilling stress (0 degree C for 3 days or at 4 degrees C for 1 week). The results suggested that isolates from Svalbard highly adapted to the severe spring condition in Polar environments.

Antagonism of Nutrient-Activated Conidia of Trichoderma harzianum (atroviride) P1 Against Botrytis cinerea.

ABSTRACT The effect of preliminary nutrient activation on the ability of conidia of the antagonist Trichoderma harzianum (atroviride) P1 to suppress Botrytis cinerea was investigated in laboratory, greenhouse, and field trials. Preliminary nutrient activation at 21 degrees C accelerated subsequent germination of the antagonist at temperatures from 9 to 21 degrees C; at >/=18 degrees C, the germination time of preactivated T. harzianum P1 conidia did not differ significantly from that of B. cinerea. When coinoculated with B. cinerea, concentrated inocula of preactivated but ungerminated T. harzianum P1 conidia reduced in vitro germination of the pathogen by >/=87% at 12 to 25 degrees C; initially quiescent conidia achieved this level of suppression only at 25 degrees C. Application of quiescent T. harzianum P1 conidia to detached strawberry flowers in moist chambers reduced infection by B. cinerea by >/=85% at 24 degrees C, but only by 35% at 12 degrees C. Preactivated conidia reduced infection by >/=60% at 12 degrees C. Both quiescent and preactivated conidia significantly reduced latent infection in greenhouse-grown strawberries at a mean temperature of 19 degrees C, whereas only preactivated conidia were effective in the field at a mean temperature of 14 degrees C on the day of treatment application. An antagonistic mechanism based on initiation of germination in sufficiently concentrated inocula suggests that at suboptimal temperatures the efficacy of Trichoderma antagonists might be improved by conidia activation prior to application.

First Report of Colletotrichum acutatum in Strawberry in Norway.

Anthracnose caused by Colletotrichum acutatum J. H. Simmonds was detected in strawberry (Fragaria × ananassa Duch.) for the first time in Norway in 1999. Symptoms were found in greenhouse grown strawberries in the cultivar Korona. Symptoms were typical of strawberry anthracnose: sunken, brown, and firm lesions appeared on maturing fruits. Masses of conidia were produced in acervuli in the center of lesions. The fungus was isolated on acidified potato dextrose agar. Colonies grown on potato dextrose agar (PDA) were pale to mouse gray and became dark greenish to blackish in reverse. Conidia were formed in orange to salmon pink masses in the center of the culture. Conidia in cultures were 16.5 (13.8 to 18.8) × 4.5 (3.8 to 5) µm, and were hyaline, cylindrical, with pointed ends, and aseptate. Setae were never observed in culture or on fruits. The fungus did not form an ascigerous stage in culture. Mycelial growth rate at 25 to 26°C on PDA was 8.1 to 8.4 mm per day. Morphological characters and growth rate were in accordance with previous reports on C. acutatum (1,2). The isolated fungus was confirmed to be C. acutatum by both the International Mycological Institute, Egham, England, and Centraalbureau voor Schimmelcultures, Baarn, the Netherlands. Koch's postulates were fulfilled by inoculating ripe and unripe fruits on strawberry plants with the isolated fungus. Fruits were either sprayed with a conidial suspension (106 conidia per ml) or slightly wounded with a needle that had been dipped in a conidial mass from a pure culture of C. acutatum. Symptoms appeared after 4 days at 20°C, and after 5 days, brown, sunken, circular lesions reached a size of 1 cm in diameter on wounded, ripe fruits. In unripe fruits the lesions developed more slowly, and in unwounded fruits sprayed with a conidial suspension, large, irregular spots developed. Leaves were inoculated by placing a small block of agar at the base of petioles on intact strawberry plants. The tissue underneath the agar was either unwounded or slightly wounded with a needle. After 20 days (at 20 to 25°C) some necrosis developed on both unwounded and wounded petioles. No symptoms were observed in the crown tissue where the inoculated petioles were attached. The fungus was readily reisolated from both fruits and petioles, after which typical morphological characters developed in culture as described above. References: (1) P. S. Gunnell and W. D. Gubler. Mycologia 84:157, 1992. (2) B. J. Smith and L. L. Black. Plant Dis. 74:69, 1990.

Isolation and characterization of a cDNA from Trichoderma harzianum P1 encoding a 14-3-3 protein homolog.

A full-length cDNA close, Th1433, (GenBank accession No. U24158), was isolated and characterized from the filamentous fungus, Trichoderma harzianum. The deduced amino acid (aa) sequence showed an acidic 30-kDa protein homologous to the 14-3-3 proteins, a family of putative kinase regulators originally characterized in mammalian brain tissue. The greatest homology, 71% identical aa, was found to BMH1, the corresponding protein from Saccharomyces cerevisiae and to the epsilon isoform from sheep brain. Southern analysis of genomic DNA indicated that Th1433 is a member of a small genomic family. At least two genes encoding 14-3-3-like proteins exist in T. harzianum. Northern analysis showed the highest level of expression during the first day after inoculation of the culture with conidial spores.

Characterization of production and enzyme properties of an endo-beta-1,4- glucanase from Bacillus subtilis CK-2 isolated from compost soil.

Bacillus subtilis CK-2, isolated from garden organic waste compost, was found to have high hydrolytic activity against carboxymethylcellulose (CMC) due to the secretion of an endo-beta-1,4- glucanase. Enzyme production was related to the sporulation process, and was regulated by the concentration of readily metabolizable carbohydrate in growth medium. Enzyme production did not require CMC or other cellulose containing materials. The endo-beta-1,4-glucanase activity was optimal at pH 5.6-5.8 and at 65 degrees C, and achieved thermal stability up to 55 degrees C. The activity was inhibited by Hg2+. The purified enzyme gave a single band corresponding to a MW of 35.5 kDa on SDS-PAGE, while the Sephadex G-75 chromatography revealed a molecular weight of the active enzyme around 70 kDa, indicating a dimeric form of the active enzyme. The enzyme activity was irreversibly inhibited by SDS. Native PAGE and IEF revealed three different isoelectric forms of the enzyme, all with an identical N-terminal amino-acid sequence.

Nucleotide sequence of an endo-beta-1,4-glucanase gene from Bacillus subtilis CK-2.

The gene encoding endo-beta-1,4-glucanase in Bacillus subtilis CK-2 was cloned into Escherichia coli DH5 alpha, and the nucleotide sequence determined. The 1500 bp gene encodes a protein of 499 amino-acid residues with a calculated molecular mass of 55,261, and is equipped with a typical B. subtilis signal peptide. Nucleotide sequence comparison revealed only 2 basepairs deviation between this gene and the endo-beta-1,4-glucanase gene of B. subtilis PAP115, and 93% to 95% homology was found between the amino acid sequences of these enzymes and other B. subtilis endo-beta-1,4-glucanases. Regions of similarity were also observed between the carboxy-terminal part of these enzymes and the part of the B. lautus PL236 celA enzyme constituting the cellulose-binding domain.

Isolation and sequence of an endochitinase-encoding gene from a cDNA library of Trichoderma harzianum.

There are no reports of gene sequences coding for extracellular chitinolytic enzymes from filamentous fungi, even though these enzymes are considered critical to the biological control of plant pathogenic fungi. The purpose of this paper was to report the isolation of a gene (ThEn-42) encoding endochitinase (Ech) from Trichoderma harzianum strain P1, describe its sequence, and to determine whether it was related to genes coding for enzymes with similar functions from prokaryotic or other eukaryotic sources. A clone containing a 1096-bp foreign cDNA fragment was isolated from thalli grown under induced conditions. This cDNA molecule was sequenced and found to lack a portion of the 5' terminus. Polymerase chain reaction (PCR) was used to isolate a fragment from the lambda gt11 library which contained the 5' terminus plus an overlap region with the 1096-bp cDNA clone. The full-length cDNA sequence, consisting of 1554 bp, contained an open reading frame (ORF) expressing a protein of 424 amino acids (aa). Southern analysis of genomic DNA indicated that there is only a single gene in strain P1 with sequence identity to the sequence described in this report. One region within the protein, thought to be required for catalytic activity of the enzyme, was highly conserved between genes coding for Ech from Th, Serratia marcescens, Bacillus circulans, Streptomyces plicatus, Vibrio parahemolyticus and Kluyveromyces lactis.

Detection and quantification of N-acetyl-beta-D-glucosaminidase, chitobiosidase, and endochitinase in solutions and on gels.

Procedures are described for the direct assay of N-acetyl-beta-glucosaminidase (EC 3.2.1.30), chitobiosidase, and endochitinase (EC 3.2.1.14) after separation on starch or polyacrylamide electrophoresis gels. The enzymes were visualized as fluorescent bands by using an agarose overlay containing 4-methylumbelliferyl derivatives of N-acetyl-beta-D-glucosaminide, beta-D-N,N'-di-acetylchitobioside, or beta-D-N,N',N"-triacetylchitotriose for N-acetyl-beta-glucosaminidase, chitobiosidase, or endochitinase, respectively. For quantitative assay of N-acetyl-beta-glucosaminidase and chitobiosidase in solutions, a rapid technique using nitrophenyl-N-acetyl-beta-D-glucosaminide and nitrophenyl-beta-D-N,N'-diacetyl-chitobiose, respectively, was used. Endochitinase activity was quantitatively measured by determining the percentage reduction in turbidity of a reaction mixture that contained purified colloidal chitin. Trichoderma harzianum strain P1 was shown to produce three kinds of chitinolytic enzymes, and there were multiple forms of some of these.

Carbohydrate content and glycosidase activities following cold hardening in two grass species.

The freezing resistance of the grass species Phleum pratense L. (timothy) and Phalaris arundinaces L. increases significantly after cold hardening. The content and composition of soluble carbohydrates were determined in the plants after short day treatment, cold hardening and dehardening. The amounts of mono-, di- and trisaccharides were reduced during the short day treatment, increased during cold hardening and decreased again during dehardening. The total amounts of soluble carbohydrates (mono-, di-, tri- and polysaccharides) were the same in hardened and dehardened plants, indicating that during hardening soluble polysaccharides (fructose polymers, fructans) were converted to mono- and oligosaccharides. Sucrose increased most after hardening conditions and, in P. arundinacea, a significant increase in 1-F-fructosylsucrose (isokestose) was also observed. Invertase (ß-fructofuranosidase. EC 3.2.1.26) activity increased following cold hardening and decreased following dehardening, while the α-galactosidase (EC 3.2.1.22) activity seemed to increase after dehardening. The glycosidases are probably involved in the mobilisation of polysaccharides during cold hardening.