Chitinase in cucumber hypocotyls is induced by germinating fungal spores and by fungal elicitor in synergism with inducers of acquired resistance

After root pretreatment with 2,6-dichloroisonicotinic acid (DCIA or INA), hypocotyls of etiolated cucumber seedlings acquired resistance to infection by Colletotrichum lagenarium caused by the failure of the fungus to penetrate epidermal cell walls. The hypocotyls contained only low levels of class III chitinase and its mRNA prior to infection. This pathogenesis-related (PR) gene was expressed strongly upon infection but only in resistant hypocotyls and soon after germination of the fungal spores. Chitinase was also induced early by an albino mutant strain of C. lagenarium that can barely penetrate the epidermis. Thus, early recognition of the fungus implies signal compounds able to pass, or being generated in, the hydrophobic epidermal surface. As the apoplastic chitinase accumulates timely at the site of a subsequent attack, it may contribute to disease resistance. The mechanism behind the enhanced responsiveness of epidermal cells was studied by gently abrading the cuticle of susceptible hypocotyls to allow permeation of a water-soluble polymeric fungal elicitor. Induction of chitinase occurred only when the elicitor was applied simultaneously with a resistance inducer such as DCIA, salicylic acid (SA) or a benzothiadiazole (BTH). In addition, long-term root pretreatment with DCIA conditioned the hypocotyls for enhanced elicitor responses. These results demonstrate that the above inducers of acquired resistance can affect expression of the cucumber chitinase gene not only as direct inducers. They can also act synergistically with fungal elicitors and, in addition, condition the hypocotyls in a developmental manner for potentiated elicitation.     

Degradation of Uredospores of the Soybean Rust Fungus (Phakopsora pachyrhizi Syd.) by Cell-Free Culture Filtrates of the Mycoparasite Verticillium Psalliotae Treschow

Verticillium psalliotae isolates Taiw and Thai C are effective parasites of the soybean rust fungus. Cell-free culture filtrates of these fungi, prepared after growth on autoclaved uredospores, contained β-1,3-glucanase, chitinase and protease activities and caused degradations, when rust spores were treated with them for 24 or 72 h. During these lytic processes carbohydrates, amino compounds and N-acetylhexosamine were released. The carbohydrate fraction was composed of mannitol, arabitol, trehalose, glucose and unidentified substances showing low R_f-values during thin layer chromatography. The amino compounds consisted of 10 amino acids (leucine and/or isoleucine, phenylalanine, glutamic acid, valine, arginine, asparagine, glutamine, serine, aspartic acid, histidine) and 5—7 substances which could not be identified. Verticillium lecanii isolate Konz is a weak parasite of soybean rust. During growth on uredospores the fungus produced culture filtrates without chitinase activity and with low total activities of β-1,3-glucanase and protease. Compared with V. psalliotae, culture filtrates of V. lecanii exerted lower lytic activities on soybean rust uredospores. The results are consistent with the aspect that the rapid growth of V. psalliotae on soybean rust fungus is primarily based on the secretion of lytic enzymes which make nutrients available to the mycoparasite.     

Over-expression of a cacao class I chitinase gene in Theobroma cacao L. enhances resistance against the pathogen, Colletotrichum gloeosporioides

Theobroma cacao L. plants over-expressing a cacao class I chitinase gene (TcChi1) under the control of a modified CaMV-35S promoter were obtained by Agrobacterium-mediated transformation of somatic embryo cotyledons. Southern blot analysis confirmed insertion of the transgene in eight independent lines. High levels of TcChi1 transgene expression in the transgenic lines were confirmed by northern blot analysis. Chitinase activity levels were measured using an in vitro fluorometric assay. The transgene was expressed at varying levels in the different transgenic lines with up to a sixfold increase of endochitinase activity compared to non-transgenic and transgenic control plants. The in vivo antifungal activity of the transgene against the foliar pathogen Colletotrichum gloeosporioides was evaluated using a cacao leaf disk bioassay. The assay demonstrated that the TcChi1 transgenic cacao leaves significantly inhibited the growth of the fungus and the development of leaf necrosis compared to controls when leaves were wound inoculated with 5,000 spores. These results demonstrate for the first time the utility of the cacao transformation system as a tool for gene functional analysis and the potential utility of the cacao chitinase gene for increasing fungal pathogen resistance in cacao.     

Short communication: Protoplast formation from the thermophilic fungus Malbranchea sulfurea,using the thermostable chitinase and laminarinase of Paecilomyces varioti

Two thermostable enzymes produced by the thermophilic fungus Paecilomyces varioti, a chitinase and laminarinase, were used to isolate protoplasts of a thermophilic fungus, Malbranchea sulfurea. The frequency of protoplast regeneration observed (35%) was considerably higher than that obtained using commercial lytic enzymes.     

Heterologous expression of genes mediating enhanced fungal resistance in transgenic wheat.

Three cDNAs encoding the antifungal protein Ag-AFP from the fungus Aspergillus giganteus, a barley class II chitinase and a barley type I RIP, all regulated by the constitutive Ubiquitin1 promoter from maize, were expressed in transgenic wheat. In 17 wheat lines, stable integration and inheritance of one of the three transgenes has been demonstrated over four generations. The formation of powdery mildew (Erysiphe graminis f. sp. tritici) or leaf rust (Puccinia recondita f. sp. tritici) colonies was significantly reduced on leaves from afp or chitinase II- but not from rip I-expressing wheat lines compared with non-transgenic controls. The increased resistance of afp and chitinase II lines was dependent on the dose of fungal spores used for inoculation. Heterologous expression of the fungal afp gene and the barley chitinase II gene in wheat demonstrated that colony formation and, thereby, spreading of two important biotrophic fungal diseases is inhibited approximately 40 to 50% at an inoculum density of 80 to 100 spores per cm2.     

Interaction of the nematophagous fungus Duddingtonia flagrans on Amblyomma cajannense engorged females and enzymatic characterisation of its chitinase

The present work aimed to evaluate the production and the characterisation of a chitinase from nematophagous fungus Duddingtonia flagrans (AC001) and observe the interaction of this fungus on engorged females of Amblyomma cajennense under laboratory conditions. In assay A, the engorged females of A. cajennense were separated and immersed for 5 seconds in a fungal suspension of 10⁶ conidia/ml of the fungus D. flagrans and placed in Petri dishes, in the dark. In assay B, wheat bran supplemented with 1% chitin and liquid minimal medium was used [K₂HPO₄ (5.0 g/l), MgSO₄ (0.10 g/l), ZnSO₄ (0.0050 g/l), FeSO₄ (0.001 g/l) e CuSO₄ (0.50 mg/l)], as a substrate for chitinase production. To demonstrate the presence of chitinase in the crude extract obtained after the enzymatic extraction, a purification process was developed using a specific adsorption technique. The results from assay A demonstrated the interaction of the D. flagrans conidia produced from chitin-agar on engorged females of A. cajennense. In the assay B, D. flagrans produced a chitinase successfully, with a high value for enzyme activity. The molecular mass of semi-purified enzyme was estimated at approximately 34 kDa. It was concluded that the fungus produced a chitinase and has some entomopathogenic activity, as demonstrated here for the first time; however, it is strongly suggested that further studies are needed to elucidate the molecular mechanism of infection of target organisms by this fungus.     

Discovery of sporothecae in adult femaleTrochometridium Cross,with notes on analogous structures inSiteroptes Amerling (Acari: Heterostigmata)

Adult female mites of the genusTrochometridium Cross possess a pair of internal sacs between the bases of legs III and IV, which are adapted for carrying spores, apparently ascospores, of an undetermined fungus. A three-way symbiotic relationship exists between the mite, the fungus, and various bees (and possibly other holometabolous insects) which nest in relatively dry alkaline soils. The mite transports spores of the fungus to suitable sites for germination — cells of ground-nesting bees containing a bee egg or young larva which dies as a result of development of the fungus and mite. The mite may also stimulate mycelial growth, possibly by killing the young bee or by secreting a substance when feeding. The fungus provides the preferred mycelial substrate on which the mite feeds and undergoes its life cycle. This mutualistic association between the mite and fungus is at the expense of the bee, which transports the other two entities to favorable sites for their development — its newly made and provisioned cells. The sporothecae and specialized association ofTrochometridium mites with a fungal pathogen or saprophyte of another host organism are compared with those ofSiteroptes mites, which are considerably better understood.     

New acidic chitinase isoforms induced in tobacco roots by vesicular-arbuscular mycorrhizal fungi

Summary Chitinase activities have been compared in tobacco roots (Nicotiana tabacum cv. Xanthi nc) infected by the pathogenic fungus Chalara elegans or three species of vesicular arbuscular mycorrhizal (VAM) fungi: Glomus versiforme, G. intraradix and G. fasciculatum, using native polyacrylamide gel electrophoresis (PAGE). All previously known acidic chitinase isoforms were stimulated in roots by the pathogenic fungus and by the VAM fungi, while two new acidic chitinase isoforms were specifically induced in response to the endomycorrhizal association. After separation in sodium dodecyl sulphate polyacrylamide denaturing gels (SDS-PAGE) under non-reducing conditions, the estimated apparent molecular mass for these additional acidic chitinase isoforms from VAM-colonized samples was 33 kDa, compared to 30 kDa for the main activity stimulated in C. elegans-infected root extracts.     

Geosiphon pyriforme,an Endosymbiotic Association of Fungus and Cyanobacteria: the Spore Structure Resembles that of Arbuscular Mycorrhizal (AM) Fungi

The zygomycete Geosiphon pyriforme is the only known endocyanosis of a fungus. The Nostoc spp. filaments are included in photosynthetically active and nitrogen fixing, multinucleated bladders, which grow on the soil surface. The spores of the fungus are white or slightly brownish. They are about 250 μm in diameter and develop singly on hyphal ends or, less frequently, intercalarly. The wall of the spores consists of a thin innermost layer, a laminated inner layer with a thickness of about 10–13 μm, and an evanescent outer layer. The laminated layer is composed of helicoidally arranged microfibrils, and is separated from the evanescent outer layer by a thin electron-dense sublayer. Polarisation microscopy indicates the occurrence of chitin. Shape and wall ultrastructure of the Geosiphon spores and their cytoplasm resemble that of Glomus spores, but are different from that of other genera of the Glomales and Endogonales. Germination occurs by a single thick hyphal outgrowth directly through the spore wall. Like various AM forming fungi, Geosiphon pyriforme contains endocytic bacteria-like organisms, which are not surrounded by a host membrane. Our observations indicate that Geosiphon is a potential AM fungus.     

Phylogenetic position of an arbuscular mycorrhizal fungus,Acaulospora gerdemannii, and its synanamorphGlomus leptotichum, based upon 18S rRNA gene sequence

We examined the phylogenetic position of an arbuscular mycorrhizal fungus which produces two types of spore,Acaulospora gerdemannii andGlomus leptotichum, based upon the DNA sequence of the 18S rRNA gene. DNA was extracted separately from bothGlomus-like orAcaulospora-like spores and partial 5′-terminus segments of 18S rRNA gene were amplified by the PCR method. Several clones derived from each spore type were sequenced and compared. The sequences from both spore types agreed well, confirming that these morphologically different spores were formed by the same fungus. Nucleotide substitutions were found among several clones, suggesting polymorphism of the rRNA gene in glomalean fungi. Further phylogenetic analysis based upon the whole sequence of the 18S rRNA gene showed thatA. gerdemannii may be within the order Glomales but is far from the fungi that have been analyzed and probably should be in a new family.     

Molecular cloning and characterization of a pea chitinase gene expressed in response to wounding,fungal infection and the elicitor chitosan

The fungicidal class I endochitinases (E.C.3.3.1.14, chitinase) are associated with the biochemical defense of plants against potential pathogens. We isolated and sequenced a genomic clone, DAH53, corresponding to a class I basic endochitinase gene in pea, Chil. The predicted amino acid sequence of this chitinase contains a hydrophobic C-terminal domain similar to the vacuole targeting sequences of class I chitinases isolated from other plants. The pea genome contains one gene corresponding to the chitinase DAH53 probe. Chitinase RNA accumulation was observed in pea pods within 2 to 4 h after inoculation with the incompatible fungal strain Fusarium solani f. sp. phaseoli, the compatible strain F. solani f.sp. pisi, or the elicitor chitosan. The RNA accumulation was high in the basal region (lower stem and root) of both fungus challenged and wounded pea seedlings. The sustained high levels of chitinase mRNA expression may contribute to later stages of pea's non-host resistance.     

Functional expression in Beauveria bassiana of a chitinase gene from Ophiocordyceps unilateralis,an ant-pathogenic fungus

A gene encoding chitinase was cloned from Ophiocordyceps unilateralis, a Formamidae-specific fungus, collected from Sirindhorn Peat Swamp Forest, Thailand. The O. unilateralis chitinase (OuChi) full-length gene (1311 bp) encodes 436 amino acids with the first 20 amino acids as a putative signal peptide. The gene showed highest identity (78%) to Isaria farinose endochitinase. To investigate if cross-species chitinase expression also enhances fungal toxicity, the mature OuChi gene was subcloned into an Agrobacterium binary vector pPZP-bar and then transformed into Beauveria bassiana strain BCC2659. Chitinase activity was detected using 4-methylumbelliferyl-β-D-N,N′-diacetylchitobioside. The fungal transformant expressing O. unilateralis chitinase showed higher toxicity against Spodoptera exigua. These results support the hypothesis that chitinolytic enzymes are one of several ‘virulence’ factors produced by entomopathogenic fungi during host encounter.     

High-level expression of a tobacco chitinase gene in Nicotiana sylvestris. Susceptibility of transgenic plants to Cercospora nicotianae infection

Endochitinases (E.C. 3.2.14, chitinase) are believed to be important in the biochemical defense of plants against chitin-containing fungal pathogens. We introduced a gene for class I (basic) tobacco chitinase regulated by Cauliflower Mosaic Virus 35S-RNA expression signals into Nicotiana sylvestris. The gene was expressed to give mature, enzymatically active chitinase targeted to the intracellular compartment of leaves. Most transformants accumulated extremely high levels of chitinase-up to 120-fold that of non-transformed plants in comparable tissues. Unexpectedly, some transformants exhibited chitinase levels lower than in non-transformed plants suggesting that the transgene inhibited expression of the homologous host gene. Progeny tests indicate this effect is not permanent. High levels of chitinase in transformants did not substantially increase resistance to the chitin-containing fungus Cercospora nicotiana, which causes Frog Eye disease. Therefore class I chitinase does not appear to be the limiting factor in the defense reaction to this pathogen.     

Wintersweet accumulates apoplastic chitinase with a dual role in petals

Most monocotyledons like cereals accumulate antifreeze proteins in the apoplast during cold acclimation, but it is still uncertain whether dicotyledons do. Here we report the isolation and characterisation of a 33-kD apoplastic chitinase extracted from the corolla of wintersweet (Chinmonanthus praecox communis L.), which was purified using successive column chromatography on Phenyl-Sepharose, DEAE-Sepharose, and CM-Sepharose. Antifreezing activity of chitinase was confirmed in terms of the formation of bipyramidal ice crystals and high thermal-hysteresis values. Interestingly, chitinase was also found to affect germination of fungal spores of four major plant pathogens. From these data, we hypothesize that, under natural conditions, wintersweet as one of the overwintering dicotyledons also accumulates apoplastic antifreeze proteins like monocotyledons. To our knowledge, this is the first report on the isolation of dicotyledon apoplastic chitinase with high-level antifreeze and antifungal activities.     

Chitinolytic activity of the anaerobic rumen fungus Piromyces communis OTS1

The anaerobic rumen fungus, Piromyces communis OTS1, was isolated from a fistulated goat. Its chitinolytic activity in the supernatant of media containing different types of chitin was studied. The fungus grew well in our basal medium, with and without colloidal chitin and chitin powder. N-Acetyl--glucosaminidase activity was not detected in any of the culture media. Chitinase activity, however, was detected in the basal medium with and without colloidal chitin and chitin powder. The extracellular chitinase concentrated from the fungal culture's supernatant by ammonium sulfate (80% saturation) showed highest activity at 40°C and at pH 5.5. In the other cell fractions of P. communis OTS1, N-acetyl--glucosaminidase was not detected, but chitinase activity was detected by 4-methylumbelliferyl reagents. Thus, it was found that the rumen fungus P. communis OTS1 has chitinase activity. Chitinases from the extracellular, cytosolic, and the microsomal fractiòns were mainly of the endo type of chitinase activity.     

不同基质多代培养对蚧虫病原真菌蜡蚧霉致病力的影响

【目的】研究昆虫病原真菌蜡蚧霉Lecanicilliurn lecanii(Zimmermann.)菌株No.V3.4504在不同培养基上继代培养,对菌种的菌落生长特性、胞外酶活力和对蚧虫致病力的影响。【方法】试验菌种蜡蚧霉菌株No.V3.4504是从染病蚧虫上分离的。试验蚧虫是沙里院褐球蚧Rhodococcus sariuoni Borchsenius和日本龟蜡蚧Ceroplastes japonicus Green。采用7种培养基继代培养多代。观察菌落形态特征、测定生长速率、产孢量、胞外蛋白酶和几丁质酶活性及对蚧虫的致死率。【结果】在PDA培养基上,菌落生长速率最快,但产孢量最低,胞外蛋白酶和几丁质酶的活性均呈逐代下降趋势,对两种蚧虫致死率也最低;增加蛋白胨对改善菌种致病力没有明显效果;在增加蚧虫尸体的D、E、F培养基上,菌落生长速率虽然较慢,但产孢量上升为8.83×106-9.13×106孢子/cm2。蛋白酶和几丁质酶的活性平均达到2.16-2.13 U/g和1.01-1.03 U/g,对两种蚧虫的致死率分别在55%-58%和39%-42%;在活蚧虫上连续培养3代,蛋白酶和几丁质酶的活性最高,为3.08-2.92 U/g和1.45-1.42 U/g,是PDA培养基上的1.6倍。对两种蚧虫的致死率也最高,分别达到71.30%和58.89%。蛋白酶和几丁质酶的活性与蚧虫死亡率呈正直线相关关系。【结论】采用PDA培养基连续多代培养会引起菌株No.V3.4504明显退化;在培养基中加入蚧虫尸体,对于保持菌种活力有明显效果;在活蚧虫体上继代培养对复壮菌种,提高菌种毒力的效果最佳。     

Purification and Characteristics of an Autolytic Chitinase of Piromyces communis OTS1 from Culture Medium

An autolysis chitinase was purified from the cultural medium of the anaerobic fungus Piromyces communis OTS1 by ammonium sulfate precipitation, affinity chromatography with regenerated chitin, chromato-focusing, gel filtration, and chromato-focusing again. The optimal pH and temperature were 6.0 and 50°C, respectively, for a 20-min assay. The chitinase was stable from pH 6.0 to 8.0, but was unstable at 70°C for 20 min. The molecular mass of chitinase was estimated by SDS-PAGE to be 44.9 kDa, and its pI was 4.4. The enzyme activity, which was of the ‘endo’ type, was inhibited by Hg²⁺ and allosamidin. The chitinase hydrolyzes chitin powder and fungal cell walls at a higher rate than an artificial chitin substrate. It can be concluded that extracellular chitinase is similar to cytosolic chitinase, but they are not the same protein. Received: 3 December 1996 / Accepted: 28 January 1997     

Purification and Characteristics of Membrane-Bound Chitinase of Anaerobic Ruminal Fungus Piromyces communis OTS1

A membrane-bound chitinase from cell wall fractions of the anaerobic ruminal fungus, Piromyces communis OTS1, was purified by affinity chromatography, gel filtration, and chromatofocusing. The molecular size of the chitinase was estimated by gel filtration to be 42.4 kDa and by SDS-PAGE to be 44.8 kDa, and its pI was 4.4. Activity was inhibited by Hg²⁺ and allosamidin. The activity at 39°C was greatest at pH 6.0. It had an ‘endo’ type action. Solubilization tests indicated that plasmalemma-bound chitinase was held in place by an electrostatic type interaction. Characterization of the membrane-bound chitinase was more similar to that of extracellular chitinase than cytosolic chitinase. This suggested that membrane-bound chitinase was the origin of extracellular chitinase. Received: 15 October 1997 / Accepted: 13 January 1998     

Taxol,an anticancer drug produced by an endophytic fungus <Emphasis Type="Italic">Bartalinia robillardoides</Emphasis> Tassi,isolated from a medicinal plant, <Emphasis Type="Italic">Aegle marmelos</Emphasis> Correa ex Roxb.

Taxol is an important anticancer drug widely used in the clinic. An endophytic fungus Bartalinia robillardoides (strain AMB-9) was isolated from Aegle marmelos, a medicinal plant and screened for taxol production. The fungus was identified based on the morphology of the fungal culture and the characteristics of the spores. This fungus was grown in MID liquid medium and analyzed chromatographically and spectrometrically, for the presence of Taxol. The amount of taxol produced by this endophytic fungus was quantified by HPLC. It produced 187.6 μg/L of taxol which suggests that the fungus can serve as a potential material for genetic engineering to improve the production of Taxol. This fungal taxol isolated from the organic extract of this fungal culture, has strong cytotoxic activity towards BT 220, H116, Int 407, HL 251 and HLK 210 human cancer cells in vitro, tested by Apoptotic assay.     

Chitinases of <Emphasis Type="Italic">Coffea arabica</Emphasis> genotypes resistant to orange rust <Emphasis Type="Italic">Hemileia vastatrix</Emphasis>

Two Coffea arabica — Hemileia vastatrix incompatible interactions (I₁: coffee cv. Caturra — rust race VI and I₂: coffee cv S4 Agaro — rust race II) and a compatible interaction (coffee cv. Caturra — rust race II) were compared in relation to the infection process and chitinase activity. In the two incompatible interactions the fungus ceased growth in the early infection stages, while in the compatible interaction no fungus growth inhibition was observed. A high constitutive level of chitinase activity was detected in the intercellular fluid of healthy leaves. Upon infection, chitinase isoforms were more abundant in incompatible interactions than in the compatible interaction. Immunodetection showed that class I chitinases are particularly relevant in the incompatible interactions and might participate in the defence response of the coffee plants.