Expression of a rice <Emphasis Type="Italic">GLP</Emphasis> in <Emphasis Type="Italic">Medicago truncatula</Emphasis> exerting pleiotropic effects on resistance against <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> through enhancing FeSOD-like activity

To evaluate the effectiveness of a germin-like protein (GLP) in legumes against the serious soil-borne pathogen Fusarium oxysporum f. sp. lentis, an Oryza sativa root-expressed GLP (OsRGLP1) was expressed in the model legume Medicago truncatula using the recombinant vector pCOsRGLP1. The transgene was highly expressed in M. truncatula transformed lines as assessed by RT-qPCR. Consistent with the active status of the transgene there was an elevated accumulation of H₂O₂ in transformed progeny. Enzymatic characterization of T₁ transgenic progeny showed increased superoxide dismutase (SOD) activity. The additional SOD activity in transgenic lines was insensitive to potassium cyanide and sensitive to H₂O₂ indicating its resemblance to FeSOD. The effectiveness of the OsRGLP1 gene was tested by monitoring the root disease after infection of wild-type and transgenic lines. Wild-type plants were greatly affected by the pathogen infection showing a percent disease index value of 50 compared to 10–18 for the transgenic lines. The tolerance of the transgenic lines leads to recovery in fresh weight and pod production to an almost normal level. Analysis of defense-related genes downstream of hydrogen peroxide (H₂O₂) in transgenic plants showed induction of salicylic acid and jasmonate signaling pathways and increased expression of some pathogenesis-related-1 (PR-1) genes and a plant defensin gene. Overall, the findings suggest that OsRGLP1 provides protection against the fungal pathogen F. oxysporum that may involve the direct influence of H₂O₂ on signaling pathways leading to the activation of defense-related genes.     

RAPD based genetic diversity among different isolates of <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp<Emphasis Type="Italic">. lycopersici</Emphasis> and their comparative biocontrol

Fusarium wilt of tomato (Solanum lycopersicum Mill.) caused by Fusarium oxysporum f. sp. lycopersici (Sacc.) W. C. Snyder and H. N. Hans (Fol.), is most serious and versatile pathogen. Chemical control of disease is not satisfactory and biological control is an attractive and potential alternative to the use of chemicals to control fusarium wilt of tomato. No any bioagent is universally effective everywhere therefore, search for potential biocontrol agent is continuous process and mandatory for several and individual ecological niches. In this experiment biocontrol efficacy of five species of Aspergillus and five species of Trichoderma were evaluated in vitro against Fusarium oxysporum f. sp. lycopersici. In both the experiments (dual culture and culture filtrates) T. harzianum was found to be highly effective against the isolates of Fol. followed by A. niger biocontrol potential of A. terreus is least among all the isolates tested. Culture filtrates obtained from A. luchuensis exerted least inhibition of Fol. The most sensitive isolate of Fol. against all the antagonists tested was identified as IIVR-2 (Fol. 9). Inherent diversity among Fol. isolates, from different tomato growing regions in India, was determined using RAPD primers. The genetic similarity coefficients ranged from 0.20 to 0.96, indicating that no any two or more isolates were 100% similar. RAPD profiles revealed up to 20% genetic diversity among ten isolates of Fusarium oxysporum f. sp. lycopersici.     

<Emphasis Type="Italic">Fusarium sambucinum</Emphasis> isolate FS-94 induces resistance against fusarium wilt of tomato via activation and priming of a salicylic acid-dependent signaling system

The wheat rhizosphere-inhabiting nonpathogenic Fusarium sambucinum isolate FS-94 protected tomato from Fusarium wilt (F. oxysporum f. sp. lycopersici) in laboratory experiments. Seed soaking or immersion of seedling roots in a FS-94 spore suspension prior to inoculation with the pathogen delayed the appearance of wilt symptoms and significantly reduced disease severity in plants of a susceptible tomato cultivar. Quantification of fungal ergosterol in infected tomato showed that protection against wilt agent was related to limitation of the pathogen growth in plants exposed to FS-94. Incubation of tomato seedlings in a FS-94 spore suspension for 48 or 72 h led to plant protection and increased the salicylic acid (SA) concentration in their roots, suggesting that this isolate was involved in a plant-mediated mode of action and induced resistance. Soaking tomato seeds in the spore suspension did not induce SA accumulation in seedling roots, but nevertheless resulted in a significant reduction in wilt severity when the seedlings were challenged with the pathogen. In response to pathogen attack, the SA content in susceptible seedlings grown from FS-94-treated seeds started to increase within 1 day and remained elevated for 72 h. This suggests that F. sambucinum isolate FS-94 primed a SA-dependent signaling system in tomato.     

Pleiotropic effect of the insertion of the <Emphasis Type="Italic">Agrobacterium rhizogenes rolD</Emphasis> gene in tomato (<Emphasis Type="Italic">Lycopersicon esculentum</Emphasis> Mill.)

The Agrobacterium rhizogenes rolD gene, coding for an ornithine cyclodeaminase involved in the biosynthesis of proline from ornithine, has been inserted in Lycopersicon esculentum cv Tondino with the aim of studying its effects on plant morphological characters including pathogen defense response. The analysis of plants transgenic for rolD did not show major morphological modifications. First generation transgenic plants however were found to flower earlier, and showed an increased number of inflorescences and higher fruit yield. Transformed plants were also analysed for parameters linked to pathogen defense response, i.e. ion leakage in the presence of the toxin produced by the fungus Fusarium oxysporum f. sp. lycopersici, and expression of the pathogenesis-related PR-1 gene. All the plants harbouring the rolD gene were shown to be more tolerant to the toxin in ion leakage experiments, with respect to the untransformed regenerated controls and the cv Tondino. PR-1 gene expression was quantitated by means of real-time PCR both at the basal level and after treatment with salicylic acid, an inducer of Systemic Acquired Resistance. In both cases the amount of PR-1 mRNA was higher in the transgenic plants. It seems therefore that the transformation of tomato plants with rolD could lead to an increased competence for defense response, as shown by toxin tolerance and increased expression of the Systemic Acquired Resistance marker gene PR-1. The results are finally discussed in view of their possible economic relevance.Communicated by G. Wenzel     

Reactive oxygen species (ROS) and antioxidative enzyme status in <Emphasis Type="Italic">Solanum lycopersicum</Emphasis> on priming with fluorescent <Emphasis Type="Italic">Pseudomonas</Emphasis> spp. against <Emphasis Type="Italic">Fusarium oxysporum</Emphasis>

In plants, ROS signaling and increase in activities of antioxidants are among defense responses. The present study describes the oxidative stress profiling in model host plant tomato (Solanum lycopersicum L.), during an invasion of the wilt pathogen Fusarium oxysporum f. sp. lycopersici with or without seed priming with Pseudomonas isolates M80, M96 and T109. Tomato seeds were primed with known Pseudomonas isolates M80, M96 and T109 and the forty-day- old plants were challenged with spores of F. oxysporum under greenhouse conditions. Leaf samples were collected at 0, 24, 48 72 and 96 h post fungal challenge and analysed for systemic level of oxidative stress parameters including total phenolics, proline, hydrogen peroxide, lipid peroxidation and enzymatic antioxidants. Disease incidence in the plants under greenhouse conditions was also calculated. Results revealed that priming with Pseudomonas isolates resulted in reduced oxidative stress in the host, during pathogen invasion. M80-priming showed highest antioxidative protection to the host plants during F. oxysporum invasion. The observed reduction in hydrogen peroxide and lipid peroxidation in primed plants was in agreement with the increased activities of the corresponding antioxidant enzymes. Greenhouse results showed that the highest wilt disease symptoms were with M80-priming followed by M96 and T109. The present study gives substantial evidences on the oxidative stress mitigation in response to Pseudomonas-priming on the model tomato-Fusarium interaction system.     

Factors effecting chitinase activity of <Emphasis Type="Italic">Rhizobium</Emphasis> sp. from <Emphasis Type="Italic">Sesbania sesban</Emphasis>

Twenty six Rhizobium strains isolated from root nodules of Sesbania sesban were studied for chitinase activity on chitin agar plates. Among them, only 12 strains showed chitinase activity. The strain showing the highest chitinase activity was selected based on maximum clear zone/colony size ratio on chitin agar plates and chitinase activity in culture filtrate. The strain was identified as Rhizobium sp. which showed a high degree of similarity with Rhizobium radiobacter (= Agrobacterium radiobacter). The cultural and nutritional conditions were optimized for maximum chitinase activity. The Rhizobium sp. exhibited maximum chitinase activity after 36 h of incubation, at neutral pH. Among the different nutritional sources, arabinose and yeast extract were found to be good inducers for chitinase activity. Rhizobium sp. could degrade and utilize dead mycelia of Aspergillus flavus, Aspergillus niger, Curvularia lunata, Fusarium oxysporum and Fusarium udum.     

<Emphasis Type="Italic">Aeromonas caviae</Emphasis> CB101 Contains Four Chitinases Encoded by a Single Gene <Emphasis Type="Italic">chi1</Emphasis>

Aeromonas caviae CB101 secretes four chitinases (around 92, 82, 70, and 55 kDa) into the culture supernatant. A chitinase gene chi1 (92 kDa) was previously studied. To identify the genes encoding the remaining three chitinases, a cosmid library of CB101 was constructed to screen for putative chitinase genes. Nine cosmid clones were shown to contain a chitinase gene on chitin plates. Surprisingly, all the positive clones contained chi1. In parallel, we purified the 55-kDa chitinase (Chi55) from the CB101 culture supernatant by continuous DEAE-Sepharose and Mono-Q anion exchange chromatography. The N-terminal amino acid sequence of the purified chitinase exactly matched the N-terminal sequence of mature Chi1, indicating that the purified chitinase (Chi55) is a truncated form of Chi1. The N- and C-terminal domains of chi1 were cloned, expressed, and purified, separately. Western blots using anti-sera to the N- and C-terminal domains of chi1 on the chitinases of CB101 showed that the four chitinases in the culture supernatant are either chi1 or C-terminal truncations of Chi1. In addition, the CB101 chi1 null mutant showed no chitinolytic activity, while CB101 chi1 null mutant complemented by pUC19chi1 containing chi1 showed all four chitinases in gel activity assay. These data indicated that all four chitinases secreted by CB101 in the culture supernatant are the product of one chitinase gene chi1.     

Irregular growth forms and cell wall modifications,polygalacturonase detection,and endocell formation in <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">radicis-lycopersici</Emphasis> infecting tomato plants,as studied ultrastructurally and cytochemically

Pathogen cells of Fusarium oxysporum f.sp. radicis-lycopersici infecting container-grown tomato plants were characterized ultrastructurally, using gold-complexed probes, chitinase and wheat germ agglutinin to localize chitin, and polyclonal antibodies to a polygalacturonase to localize this enzyme. It was isolated and purified from the pathogen growing in culture. Many fungal cells were of irregular forms (microhyphal, frondose) with modified, thin or imperceptible lucent wall layers, in which were often included components seemingly of host origin. Gold particles of the polygalacturonase probe were concentrated on portions of penetration hyphae and in areas of associated altered host wall. Fine filamentous-like structures, often linked to fungal cells, reached into extracellular matter and into host walls. Examination of 0.2–0.25 μm-thick sections at 120 kV, and tilted at various angles, indicated that fungal cells frequently had a pronounced wavy contour. Labelling of thin walls for chitin was mostly nil, particularly in contact with host walls, as of also thicker walls in similar situations, or it was then associated with the outside opaque layer. Cells of diverse dimensions with thin or thicker walls and with altered or normal content, contained endocells. Walls of the encodcells and of the enclosing cells often labelled differently for chitin with both probes. Endocells mostly did not originate from proliferation of a living into a dead cell but often ensuing as an apparent fragmentation of the cell content or following its retraction. The bearing of these observations on the host-pathogen relationship, particularly concerning the role of thin-walled hyphae and irregular forms, is discussed.     

Chitinase isozymes induced by TYMV and <Emphasis Type="Italic">Leptosphaeria maculans</Emphasis> during compatible and incompatible interaction with <Emphasis Type="Italic">Brassica napus</Emphasis>

Accumulation of extracellular chitinases in Brassica napus plants infected with Turnip yellow mosaic virus (TYMV) and fungal pathogen Leptosphaeria maculans was studied in both compatible and incompatible interaction. Analysis of apoplast fluid by means of non-denaturing anodic and cathodic PAGE followed by in-gel detection of chitinase activity revealed a number of chitinase isozymes. TYMV induced 8 acidic and 4 basic isozymes in a systemic way. Except for one acidic and one basic isozyme, all other chitinases were also constitutively present in low amounts in mock inoculated control. In TYMV systemically infected plants, chitinases were detected in leaves expressing symptoms as well as in symptomless ones. Both virulent and avirulent L. maculans isolates induced production of chitinase isozymes in cotyledons in a time dependent manner. Some of them were present in plants constitutively and their content increased after inoculation. Three of five acidic and two of three basic isozymes responded to L. maculans infection. Chitinases started to accumulate before symptom appearance. First two acidic isozymes were detected 24 h after inoculation. The difference between compatible and incompatibe interaction reflected two basic isozymes.     

Combined expression of a barley class II chitinase and type I ribosome inactivating protein in transgenic <Emphasis Type="Italic">Brassica juncea</Emphasis> provides protection against <Emphasis Type="Italic">Alternaria brassicae</Emphasis>

Alternaria leaf spot caused by Alternaria brassicae, or A. brassicola, is one of the major fungal diseases of Brassica juncea (Indian mustard). To develop resistance against this fungal disease, the barley antifungal genes class II chitinase (AAA56786) and type I ribosome inactivating protein (RIP; AAA32951) were coexpressed in Indian mustard via Agrobacterium-mediated transformation. The stable integration and expression of transgenes in T₀ plants were confirmed by Southern blot and Western analysis. The transgenic lines showing inheritance in Mendalian fashion (3:1) were further evaluated by in vitro studies and under greenhouse conditions for resistance to the A. brassicae fungal pathogen. The transgenic plants showed up to 44% reduction in A. brassicae hyphal growth in in vitro antifungal assays. In green house screening, the transgenic plants sprayed with A. brassicae spores showed resistance through delayed onset of the disease and restricted number, size, and expansion of lesions as compared to wild type plants. These results indicate that the expression of chitinase and RIP from a heterologous source in B. juncea provide subsequent protection against Alternaria leaf spot disease and can be helpful in increasing the production of Indian mustard.     

Transgenic rice plants expressing trichothecene 3-<Emphasis Type="Italic">O</Emphasis>-acetyltransferase show resistance to the <Emphasis Type="Italic">Fusarium</Emphasis> phytotoxin deoxynivalenol

Fusarium head blight (FHB) is a devastating disease of small grain cereal crops caused by the necrotrophic pathogen Fusarium graminearum and Fusarium culmorum. These fungi produce the trichothecene mycotoxin deoxynivalenol (DON) and its derivatives, which enhance the disease development during their interactions with host plants. For the self-protection, the trichothecene producer Fusarium species have Tri101 encoding trichothecene 3-O-acetyltransferase. Although transgenic expression of Tri101 significantly reduced inhibitory action of DON on tobacco plants, there are several conflicting observations regarding the phytotoxicity of 3-acetyldeoxynivalenol (3-ADON) to cereal plants; 3-ADON was reported to be highly phytotoxic to wheat at low concentrations. To examine whether cereal plants show sufficient resistance to 3-ADON, we generated transgenic rice plants with stable expression and inheritance of Tri101. While root growth of wild-type rice plants was severely inhibited by DON in the medium, this fungal toxin was not phytotoxic to the transgenic lines that showed trichothecene 3-O-acetylation activity. This is the first report demonstrating the DON acetylase activity and DON-resistant phenotype of cereal plants expressing the fungal gene. S. Ohsato and T. Ochiai-Fukuda should be considered as joint first authors.     

Biochemical and genetic analysis of a unique poly(ADP-ribosyl) glycohydrolase (PARG) of the pathogenic fungus <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">lycopersici</Emphasis>

The genome sequence of the plant pathogen Fusarium oxysporum f. sp. lycopersici contains a single gene encoding a predicted poly(ADP-ribose) glycohydrolase (FOXG_05947.2, PARG). Here, we assessed whether this gene has a role as a global regulator of DNA repair or in virulence as an ADP ribosylating toxin homologue of bacteria. The PARG protein was purified after expressing its encoding gene in Escherichia coli. Its inhibition by 6,9-diamino-2-ethoxyacridine lactate monohydrate and tannins was similar to its human orthologue that is involved in DNA repair. A deletion strain of F. oxysporum f. sp. lycopersici showed no growth defects and was not affected in pathogenicity. Together, our results indicate that the PARG protein of F. oxysporum f. sp. lycopersici is involved in DNA repair and does not act in pathogenicity as an effector.     

Transgenic <Emphasis Type="Italic">Brassica napus</Emphasis> L. lines carrying a two gene construct demonstrate enhanced resistance against <Emphasis Type="Italic">Plutella xylostella</Emphasis> and <Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis>

Plant diseases and insect pests are serious threat to the growth and yield of oilseed rape. In this study, a binary vector carrying sporamin and chitinase PjChi-1 genes in tandem was introduced into Brassica napus cv. ZS 758 via Agrobacterium tumefaciens for dual resistance against disease and insect attack. Thirty-two regenerated plantlets exhibiting hygromycin resistance were selected following Agrobacterium-mediated transformation of 600 leaf petiole explants. Of these, 27 transformants were confirmed to carry the two transgenes as detected by polymerase chain reaction (PCR) with 4.5% transformation efficiency. Eight plantlets were randomly selected for further confirmation by Southern and northern blot hybridization analyses. Four plants carried single copy of the transgenes, while the remaining four plants carried either two or three copies of the transgenes. Moreover, expression of the sporamin transgene was detected by northern blot hybridization in transgenic lines, but not in wild-type plants. These eight T₀ plants were grown in vitro, and inoculated with the Lepidoptera larvae of Plutella xylostella and with spores of the fungal pathogen of Sclerotinia sclerotiorum. Transgenic plants exhibited high levels of resistance to P. xylostella and S. sclerotiorum when compared to untransformed wild-type plants. Genetic analysis of T₁ progeny confirmed Mendelian segregation of the introduced genes. Therefore, these transgenic lines demonstrate a promising potential for variety development of oilseed rape lines with enhanced resistance against both P. xylostella and S. sclerotiorum.     

Antifungal Activity of Chitinases from <Emphasis Type="Italic">Trichoderma aureoviride</Emphasis> DY-59 and <Emphasis Type="Italic">Rhizopus microsporus</Emphasis> VS-9

Two chitinolytic fungal strains, Trichoderma aureoviride DY-59 and Rhizopus microsporus VS-9, were isolated from soil samples of Korea and Vietnam, respectively. DY-59 and VS-9 crude chitinases secreted by these fungi in the 0.5% swollen chitin culture medium had an optimal pH of 4 and the optimal temperatures of 40°C and 60°C, respectively. Enzymatic hydrolysis products from crab swollen chitin were N-acetyl-β-D-glucosamine (GlcNAc) by DY-59 chitinase, and GlcNAc and N, N′-diacetylchitobiose (GlcNAc)₂ by VS-9 chitinases. The chitinases degraded the cell wall of Fusarium solani hyphae to produce oligosaccharides, among which GlcNAc, (GlcNAc)₂, and pentamer (GlcNAc)₅ were identified by high-pressure liquid chromatography. DY-59 and VS-9 chitinases inhibited F. solani microconidial germination by more than 70% and 60% at final protein concentrations of 5 and 27 μg mL⁻¹, respectively, at 30°C for 20 h treatment.     

Stability of sheath blight resistance in transgenic ASD16 rice lines expressing a rice <Emphasis Type="Italic">chi11</Emphasis> gene encoding chitinase

Development of transgenic plants by introducing defense genes is one of the strategies to engineer disease resistance. Transgenic ASD16 rice plants harbouring rice chitinase chi11 gene, belonging to a PR-3 group of defense gene conferring sheath blight (Rhizoctonia solani Kuhn) resistance, were used in this study. Three T₂ homozygous lines (ASD16-4-1-1, 5-1-1, and 6-1-1) were identified from seven putative (T₀) transgenic lines expressing chi11 using Western blotting analysis. The inheritance of sheath blight resistance in those lines was studied over generations. The stability of chi11 expression up to T₄ generation in all the three homozygous lines was proved by Western blot and the stability of sheath blight resistance in the homozygous lines was proved up to T₄ generation using detached leaf and intact leaf sheath assays. Among the three homozygous lines tested, ASD16-4-1-1 showed consistent results in all the generations and gave a better protection against the sheath blight pathogen than the other two lines.     

Aluminium pre-treatment induces activation of defense responses against <Emphasis Type="Italic">Fusarium</Emphasis> infection in <Emphasis Type="Italic">Triticum aestivum</Emphasis>

Pre-treatment with low dose of aluminium sulphate [Al₂(SO₄)₃] was assayed on wheat (Triticum aestivum L.) seedlings for its ability to induce resistance against Fusarium oxysporum infection. Pre-treatment of seven days old germinated wheat seedlings with 50 μM concentration of Al₂(SO₄)₃ reduced the severity of the disease. In Al₂(SO₄)₃ pre-treated seedlings inoculated with fungus reduction in disease severity was correlated with suppression of fungal mycelia development inside the leaf tissues. The activities of peroxidase, polyphenol oxidase, and phenylalanine ammonia lyase were determined as resistance markers. F. oxysporum inoculation induced significant increase of all these enzymes. Such responses were expressed earlier and with much higher magnitude when Al₂(SO₄)₃ pre-treated seedlings were challenged with the pathogen. Slower disease development in Al₂(SO₄)₃ pre-treated seedlings might be due to increased deposition of total phenolic compounds and enhanced level of salicylic acid which restricts pathogen entry.     

Chitinolytic and antifungal activity of a <Emphasis Type="Italic">Bacillus pumilus</Emphasis> chitinase expressed in <Emphasis Type="Italic">Arabidopsis</Emphasis>

The Bacillus pumilus SG2 chitinase gene (ChiS) and its truncated form lacking chitin binding (ChBD) and fibronectin type III (FnIII) domains were transformed to Arabidopsis plants and the expression, functionality and antifungal activity of the recombinant proteins were investigated. Results showed that while the two enzyme forms showed almost equal hydrolytic activity toward colloidal chitin, they exhibited a significant difference in antifungal activity. Recombinant ChiS in plant protein extracts displayed a high inhibitory effect on spore germination and radial growth of hyphae in Alternaria brassicicola, Fusarium graminearum and Botrytis cinerea, while the activity of the truncated enzyme was strongly abolished. These findings demonstrate that ChBD and FnIII domains are not necessary for hydrolysis of colloidal chitin but play an important role in hydrolysis of chitin–glucan complex of fungal cell walls. Twenty microgram aliquots of protein extracts from ChiS transgenic lines displayed strong antifungal activity causing up to 80% decrease in fungal spore germination. This is the first report of a Bacillus pumilus chitinase expressed in plant system.     

Transgenic rice plants expressing the snowdrop lectin gene (<Emphasis Type="Italic">gna</Emphasis>) exhibit high-level resistance to the whitebacked planthopper (<Emphasis Type="Italic">Sogatella furcifera</Emphasis>)

Transgenic rice plants, expressing snowdrop lectin [Galanthus nivalis agglutinin (GNA)], obtained by Agrobacterium-mediated genetic transformation, were evaluated for resistance against the insect, the whitebacked planthopper (WBPH). The transgene gna was driven by the phloem-specific, rice-sucrose synthase promoter RSs1, and the bar was driven by the CaMV 35S promoter. In our previous study, the transgenic status of these lines was confirmed by Southern, Northern and Western blot analyses. Both the transgenes, gna and bar, were stably inherited and co-segregated into progenies in T₁ to T₅ generations. Insect bioassays on transgenic plants revealed the potent entomotoxic effects of GNA on the WBPH. Also, significant decreases were observed in the survival, development and fecundity of the insects fed on transgenic plants. Furthermore, intact GNA was detected in the total proteins of WBPHs fed on these plants. Western blot analysis revealed stable and consistent expression of GNA throughout the growth and development of transgenic plants. Transgenic lines expressing GNA exhibited high-level resistance against the WBPH. As reported earlier, these transgenics also showed substantial resistance against the brown planthopper and green leafhopper .