Molecular Monitoring of hrpB-disrupted Mutant of Ralstonia solanacearum in Tomato Plants

A nonpathogenic mutant of Ralstonia solanacearum was produced by the insertion of transposon Tn4431. The mutagenized gene was then cloned from a genomic DNA library by the gene tagging method, using the labeled lux operon located on Tn4431 of pUCD623 as a hybridization probe. From nucleotide sequence analysis of the transposon-inserted genomic clone, the hrpB gene was shown to be disrupted by the inserted transposon. Tomato plants were inoculated with the hrpB-disrupted mutant bacteria, for which multiplication and translocation were then monitored using the colony hybridization method. In addition, the original pathogenic bacteria in which the lux operon had been functionally ligated with the genomic promoter were also used for inoculation and traced by their bioluminescence. Multiplication of the hrpB-disrupted mutant was suppressed initially in the invaded root tissues and then in upper hypocotyl after translocation, suggesting that the pathogenic strain of R. solanacearum overcomes at least two steps of host responses expressed in root and hypocotyl tissues. Thus, our approach for molecular monitoring of the bacteria enabled us to precisely analyze the infection behavior of the pathogenic bacteria in planta. Received 16 April 1999/ Accepted in revised form 10 August 1999     

A Monitoring System for Green Fluorescence Protein Gene-transformed Fusarium oxysporum in Melon Seedlings

Using melon seedlings at the cotyledon stage and genetically marked fungi, a system for monitoring pathogenic and nonpathogenic Fusarium oxysporum was devised in the present study. Protoplasts were prepared from three formae speciales (melonis, radicis-lycopersici and fragariae )of F. oxysporum and transformed with a synthetic gene for green fluorescence protein. Transformants were primarily isolated in the presence of hygromycin B and then screened by the emission of bright green fluorescence. Roots of melon seedlings were inoculated with fluorescing microconidia of these fungi, and fungal infection behavior was traced. Using fluorescence microscopy, we directly observed not only the fungus at the root surface, but also the mycelia elongating in the trachea of roots. Both pathogenic and nonpathogenic fungi germinated and hyphae elongated superficially on the surface of root. Only pathogenic fungi caused root necrosis at the inoculation site. Hyphae grew within the stem to induce constriction or cracking of lower hypocotyls, then causing wilting of the seedlings. Infection behavior of genetically marked pathogenic and nonpathogenic F. oxysporum could be successfully monitored after inoculation of cotyledons of seedlings. Received 6 June 2001/ Accepted in revised form 3 August 2001     

Comparative analysis of pathogenic and nonpathogenic Fusarium oxysporum populations associated with banana on a farm in Minas Gerais,Brazil

Fusarium wilt is one of the most devastating diseases on banana. The causal agent, Fusarium oxysporum f. sp. cubense (Foc) is genetically diverse and its origin and virulence are poorly understood. In this study, pathogenic Foc isolates and nonpathogenic F. oxysporum isolates from Minas Gerais in Brazil were compared using EF‐1α and IGS sequences. This allowed the examination of the origin and evolutionary potential of Foc in a country outside the region of origin of the banana plant. Two different sequence types were found among Foc isolates. One appeared to be of local origin because it was identical to the sequence type of the largest group of nonpathogenic isolates. To explore if the ‘local’ Foc isolates had acquired pathogenicity either independently through coevolution with the host, or through horizontal gene transfer (HGT) of pathogenicity genes from other, probably introduced, Foc isolates, the presence and sequence of putative SIX effector genes were analysed. Homologues of SIX1, SIX3 and SIX8 were found. SIX1 sequences were identical and exclusively found in all pathogenic isolates, while variable ratios of sequences of multicopy gene SIX8 were found among nonpathogenic and different pathogenic isolates. This observation supports the HGT hypothesis. Horizontal transfer of genes between isolates of F. oxysporum has important implications for the development of reliable diagnostic tools and effective control measures. Full genome sequencing is required to confirm HGT and to further unravel the virulence mechanisms of forma specialis cubense.     

Characterization of Elicitor-inducible Tobacco Genes Isolated by Differential Hybridization

Inducible responses in plants against pathogen attack play a major role in resistance to disease. The defense responses are mostly associated with the expression of various kinds of inducible genes. We employed differential hybridization to isolate elicitor-inducible genes (EIGs) of tobacco (Nicotiana tabacum cv. Samsun NN) using the tobacco-fungal elicitor system. A cDNA library was constructed from tobacco leaves treated for 12 hr with hyphal wall components (HWC) prepared from Phytophthora infestans, and six EIGs were identified. Expression of all EIGs was induced after inoculation with the soybean pathogen Pseudomonas syringae pv. glycinea (nonpathogenic on tobacco) or treatment with salicylic acid, and a variety of expression patterns of EIG mRNAs was observed. Sequence analysis of EIG cDNAs revealed similarities to genes for SAR8.2 (EIG-B39 and EIG-D14), glycine-rich protein (EIG-G7), extensin (EIG-I30), acyltransferase (EIG-I24) and unknown protein (EIG-J7). Possible roles of EIG products in disease resistance are discussed. Received 30 August 2000/ Accepted in revised form 30 November 2000     

Induced resistance in tomato plants to the toxin-dependent necrotrophic pathogen Alternaria alternata

A necrotrophic pathogen, the tomato pathotype of Alternaria alternata (Aa) causes Alternaria stem canker on tomato. Its pathogenicity depends on the production of host-specific AAL-toxin. Pre-inoculation with nonpathogenic Aa or pretreatment an elicitor prepared from Aa reduced disease symptoms by the pathogen. Salicylic acid (SA)- and jasmonic acid (JA)-dependent defense responses in tomato are not involved in the resistance to the pathogen induced by nonpathogenic Aa. The results suggest that an alternative and unknown signaling pathway independent of SA- and JA-signaling might modulate the induced resistance by activating the expression of the multiple defense genes.     

Death of endemic Virgilia oroboides trees in South Africa caused by Diaporthe virgiliae sp. nov.

Numerous dead and dying individuals of the Western Cape endemic tree Virgilia oroboides (Fabaceae) were recently observed within a South African national botanical garden. Root‐rot fungi and fungi symbiotic with bark beetles (Curculionidae; Scolytinae) from diseased trees were assessed for their respective roles in V. oroboides mortality. Disease progression was also monitored over 1 year. Fungi were isolated from surface sterilized bark and root samples from diseased trees and provisionally identified using data from the internal transcribed spacer regions (ITS1, ITS2), including the 5·8S rRNA gene (ITS). Pathogenicity of selected fungi towards V. oroboides was tested under field conditions. The pathogenicity of various bark beetle‐associated Geosmithia (Hypocreales: Hypocreomycetidae) spp. from V. oroboides were similarly assessed. The only fungus consistently isolated from lesions on the roots and bark of declining V. oroboides, and never from healthy individuals, represented an undescribed Diaporthe (Diaporthales, Diaporthaceae) species that was characterized using molecular (using data from the ITS marker and part of the β‐tubulin gene, TUB), cultural and morphological characters. It is an aggressive pathogen of V. oroboides, newly described here as Diaporthe virgiliae sp. nov. Trees of all ages are susceptible to this pathogen with subsequent bark beetle attack of mature trees only. All Geosmithia spp. from beetles and/or infected trees were nonpathogenic towards V. oroboides. Diaporthe virgiliae caused a severe decline in the health of the monitored V. oroboides population over a period of only 1 year and should be considered as a significant threat to these trees.     

Involvement of Iron and Ferritin in the Potato–Phytophthora infestans Interaction

This work shows that the infection of potato (Solanum tuberosum) detached leaves by the late blight pathogen Phytophthora infestans, was drastically reduced by adding deferoxamine, an exogenous iron chelator. Reactive oxygen species in leaves inoculated with P. infestans were also reduced after adding deferoxamine. A leaf ferritin cDNA fragment was obtained by PCR and used as probe for screening a tuber cDNA library. A cDNA (named StF1) encoding the iron-storing potato ferritin was cloned. StF1 is 915 bp in length and has an open reading frame of 230 amino acids that contains the information for the mature 28 kDa subunit of potato ferritin. StF1 was used as probe in northern blot hybridizations to analyze expression of the ferritin gene. In leaves, ferritin mRNA accumulated in response to pathogen attack. In tubers, ferritin mRNA increased upon treatment with the elicitor eicosapentaenoic acid. These results suggest that iron plays a role in the potato-P. infestans interaction.     

Sensitivity to,and metabolism of,phaseollin in relation to the pathogenicity of different isolates of Botrytis cinerea to bean (Phaseolus vulgaris)

The pathogenicity of five isolates ofBotrytis cinerea to bean (Phaseolus vulgaris) was compared with their sensitivity to the phytoalexin phaseollin and their ability to metabolize phaseollin. Three of the five isolates were pathogenic to bean, as they formed spreading lesions on bean leaves; the other two isolates were considered nonpathogenic to bean, since they produced lesions limited in size or no lesions at all. The isolates were about equally sensitive to phaseollin, except for one nonpathogenic strain which was more sensitive than the other ones to higher concentrations of phaseollin. The three pathogenic isolates metabolized phaseollin to 6a-hydroxyphaseollin readily in shake cultures containing 9 μg phaseollin/ml, while nonpathogenic isolates were not able, or less able, to do so.     

Cell interactions between a nonpathogenic <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> strain and root tissues of <Emphasis Type="Italic">Eucalyptus viminalis</Emphasis>

Nonpathogenic isolates of Fusarium oxysporum can be successful antagonists of pathogenic forms of the same fungal species that commonly attacks crop plants. The characteristics that distinguish nonpathogenic from pathogenic forms are not well understood. In this study, the mode of root colonization of Eucalyptus viminalis seedlings by a nonpathogenic F. oxysporum strain is described at the ultrastructural level. Root systems of E. viminalis plants were inoculated with nonpathogenic F. oxysporum strain Fo47 in an in vitro model system. Changes in the occurrence of nonesterified and methyl-esterified pectins in colonized E. viminalis roots were evaluated by in situ immunolabeling using two monoclonal antibodies, JIM 5 and JIM 7. Modes of penetration and root colonization patterns in E. viminalis seedlings by the nonpathogenic fungus were similar to those described for pathogenic forms of F. oxysporum. However, root interactions differed in that the nonpathogenic fungus did not induce host tissue damage. No papilla-like appositions were observed in host cells in response to invading hyphae, which did not disrupt the host plasma membrane in many cases, suggesting that a biotrophic relationship was established. Root colonization by the nonpathogenic strain did not induce alteration in JIM 7 labeling of methyl-esterified pectin in E. viminalis cell walls, whereas nonesterified pectin was detected to a significantly greater extent in cell walls of roots colonized by the fungus. Pectin components decreased slightly only at points of hyphal contact with host cells. Because nonpathogenic strains utilize pectin in pure culture, host control over enzyme activity or production by the fungi may at least partly explain their compatible interactions with host tissues.     

Adverse effect of the chitinolytic enzyme PjCHI‐1 in transgenic tomato on egg mass production and embryonic development of Meloidogyne incognita

A novel chitinase gene (PjCHI‐1) isolated from Paecilomyces javanicus, a non‐nematophagous fungus, and driven by a CaMV35S promoter, was delivered into CLN2468D, a heat‐tolerant cultivar of tomato (Solanum lycopersicum). T₁ tomato plants exhibited high endochitinase activity and reduced numbers of eggs and egg masses when infected with the root‐knot nematode (RKN) Meloidogyne incognita. The eggs found in transgenic tomato had lower shell chitin contents than eggs collected from control plants. Egg masses from transgenic plants exhibited higher chitinase activity than those from control plants. Moreover, only 30% of eggs from transgenic plants were able to develop to the multi‐cell/J1 stage, compared with more than 96% from control plants. The present study demonstrated that the expression of the PjCHI‐1 chitinase gene can effectively reduce the production of egg masses and repress the embryonic development of M. incognita, presenting the possibility of a novel agro‐biotechnological strategy for preventing crop damage by RKN.     

Inhibition of crown gall formation by Agrobacterium radiobacter biovar 3 strains isolated from grapevine

Graft unions of nursery stock of grapevine (Vitis vinifera L.) collected in Japan yielded pathogenic and nonpathogenic strains of Agrobacterium. On the basis of classical diagnostic tests, a sequence analysis, and a multiplex polymerase chain reaction method previously reported, the pathogenic strain was identified as Agrobacterium tumefaciens biovar 3, whereas the nonpathogenic strains were assigned to Agrobacterium radiobacter biovar 3. Stems of tomato (Lycopersicon esculentum Mill.) seedlings were inoculated with both A. tumefaciens biovar 3 strain G-Ag-27 as a pathogen and one of the control strains isolated from grapevine or A. radiobacter biovar 2 strain K84 as competitors to assay the suppression of gall formation caused by the pathogen. In a test with a 1 : 1 pathogen/nonpathogen cell ratio, all A. radiobacter biovar 3 strains reduced gall incidence and size compared to that of the positive control inoculated only with the pathogen. Strain VAR03-1 was especially effective in reducing the incidence of gall formation on grapevine and reduced gall size by 84%–100% of those on the positive control. Many tested nonpathogenic biovar 3 strains were bacteriocinogenic, causing an inhibition zone against A. tumefaciens biovar 3 strains on YMA medium. Strain VAR03-1 was the most effective against indicator strains and appears to be a promising agent for controlling crown gall of grapevine.     

A natural two-nucleotide deletion affirms role for NADPH oxidase in pathogenicity and perithecium formation in Fusarium graminearum species complex

Members of the Fusarium graminearum species complex are known to cause head blight of wheat. A natural nonpathogenic mutant, strain Fa0233007, was found in the strain collection of the Japanese F. graminearum species complex. In addition, this strain cannot form perithecia. Complete linkage of these deficiencies in the progenies obtained from crossing a wild-type strain and the mutant strain suggested that a single genomic locus was responsible for deficiencies in both traits. The locus was mapped to chromosome 1 using sequence-tagged markers. An NADPH oxidase gene (NoxA, FGSG00739) was identified in the mapped region, and a 2-bp deletion that results in premature termination of the open reading frame was detected in the gene. Both pathogenicity and perithecium formation were recovered by FGSG00739 complementation in the mutant strain.     

Sequence variation in the two-component histidine kinase gene of Botrytis cinerea associated with resistance to dicarboximide fungicides

The complete two-component histidine kinase gene (Bos1) was sequenced from eight dicarboximide-resistant (D^R) and six-sensitive (D^S) field isolates of Botrytis cinerea. Comparisons in the predicted amino acid sequences of Bos1 showed that each two D^R isolates had a single point mutation at amino acid position 365 from an isoleucine to serine (I365S) or to an asparagine (I365N). Three D^R isolates were characterized with a change from glutamine to proline at position 369 (Q369P) as well as an asparagine to serine at the position 373 (N373S). These mutations located within the 90-amino-acid repeats of Bos1 have been reported previously. One new mutation, however, was found in the D^R isolate 65-E8. In this isolate, a null mutation at the amino acid position 1040 in the Bos1 was detected. Inoculation tests showed that this isolate was nearly nonpathogenic to cucumber. After the Bos1 gene from the sensitive isolate 38B1 was transferred into the resistant isolate 65-E8, all transformants tested were sensitive to iprodione and capable of infecting cucumber. DNA fingerprint generated by micro-satellite primed-PCR showed that isolates were not clustered based on their sensitivity to iprodione. Results from this study indicate that mutations in the Bos1 gene associated with dicarboximide resistance are diverse in B. cinerea, and the Bos1 gene is associated with virulence of B. cinerea.     

In-plant activation of root-specific expression of a cytotoxic gene disrupts the development of the root-knot nematode,Meloidogyne javanica

Plant hosts can be engineered to disrupt the development of sedentary plant-parasitic nematodes or proper functioning of the feeding sites the nematodes induce. The use of constitutive promoters to express dsRNAs or nematode inhibitor proteins may be unreliable because of possible silencing or yield penalty from continuous expression in a plant host. This ill-effect can be avoided if a root-specific, nematode-responsive promoter (NRP) is used to drive the target nematode-inhibitory message. This study used the In Plant Activation (INPACT) system to express a barstar-controlled barnase in galls of Meloidogyne javanica and assessed how the engineered tobacco lines affected the growth and development of the nematodes. Of the 11 combinations of four NRPs and the CaMV 35S promoter assessed, the AtCel1 and TobRB7 combinations activated specific expression of split β-glucuronidase (GUS) and barnase genes in and around giant cells. The same NRP combination directed expression of the barnase gene in tobacco roots also constitutively expressing the barstar gene (SPBB transgenic lines). On roots of six T₁ SPBB lines, there was up to 94% reduction in the number of galls with significantly smaller adult females compared to those on wild-type plants. Some of the females on lines SPBB4-1 and SPBB-4-2, for example, were not associated with galls. The results indicated the engineered plants disrupted M. javanica development and demonstrate the potential for controlled and localized expression of peptides, such as those that could block specific effectors, to disrupt initiation, formation, establishment, or proper functioning of feeding cells induced by damaging sedentary nematodes.     

Detoxification of α-tomatine by tomato pathogens Alternaria alternata tomato pathotype and Corynespora cassiicola and its role in infection

In tomato plants, α-tomatine, a steroidal glycoalkaloid saponin, inhibits fungal growth. Tomato pathogens that produce host-specific toxins, Alternaria alternata tomato pathotype causing Alternaria stem canker and Corynespora cassiicola causing Corynespora target spot, were investigated for sensitivity to α-tomatine. Although spore germination of A. alternata pathogenic and nonpathogenic to tomato and of C. cassiicola pathogenic to tomato was not affected by 0.1 mM α-tomatine, spore germination of C. cassiicola nonpathogenic to tomato was significantly inhibited. This result showed that A. alternata, regardless of its pathogenicity, and only the C. cassiicola pathogenic to tomato are resistant to α-tomatine. Germinating spores of A. alternata and C. cassiicola resistant to α-tomatine detoxified α-tomatine by degrading it to a less polar product. After inoculation of tomato leaves, spores of A. alternata and C. cassiicola nonpathogenic to tomato germinated and formed appressoria, but did not form infection hyphae in host tissues. When a host-specific toxin (CCT-toxin) produced by C. cassiicola pathogenic to tomato was added to nonpathogenic spores, colonization within leaves was observed in A. alternata, but not in C. cassiicola. On the other hand, when spores of C. cassiicola nonpathogenic to tomato were suspended in spore germination fluid of nonpathogenic A. alternata with α-tomatine detoxification activity, the fungus could be induced to colonize leaves in the presence of CCT-toxin. These results indicate that A. alternata tomato pathotype and C. cassiicola pathogenic to tomato detoxify α-tomatine during infection and that this detoxification is essential for host colonization by pathogens that produce host-specific toxins.