Detection of the Defoliating Pathotype of Verticillium dahliae in Infected Olive Plants by Nested PCR

Spread of Verticillium wilt into newly established olive orchards in Andalucía, southern Spain, has caused concern in the olive industry in the region. This spread may result from use of Verticillium dahliae-infected planting material, which can extend distribution of the highly virulent, defoliating (D) pathotype of V. dahliae to new areas. In this study, a molecular diagnostic method for the early in planta detection of D V. dahliae was developed, aimed especially at nursery-produced olive plants. For this purpose, new primers for nested PCR were designed by sequencing a 992-bp RAPD marker of the D pathotype. The use of the specific primers and different nested-PCR protocols allowed the detection of V. dahliae pathotype D DNA in infected root and stem tissues of young olive plants. Detection of the pathogen was effective from the very earliest moments following inoculation of olive plants with a V. dahliae pathotype D conidia suspension as well as in inoculated, though symptomless, plants.     

Gene Flow Analysis of Phytophthora porri Reveals a New Species: Phytophthora brassicae Sp. Nov.

Isozyme analysis and sequence analysis of the internal transcribed spacer regions (ITS-1 and ITS-2) and the 5.8S subunit of the ribosomal DNA gene repeat were used to examine whether isolates of Phytophthora porri from Allium and Brassica represent a single homogeneous species. Twenty-six strains of P. porri, 16 strains isolated from the genus Allium, and 10 strains isolated from the genus Brassica, were analyzed using malate dehydrogenase (MDH), isocitrate dehydrogenase (IDH) and lactate dehydrogenase (LDH), represented altogether by four putative loci (Mdh-2, Idh-1, Idh-2, and Ldh-2). Isozyme analysis revealed that strains isolated from Allium contained five private alleles at three isozyme loci (Ldh-2 ⁸³, Ldh-2 ¹⁰⁴, Idh-1 ¹⁰⁸, Idh-1 ¹¹², and Idh-2 ⁹⁸), whereas six different alleles were observed at four isozyme loci (Ldh-2 ⁸⁵, Ldh-2 ¹⁰⁰, Ldh-2 ¹¹⁴, Idh-1 ¹⁰⁰, Idh-2 ¹⁰⁰, and Mdh-2 ¹¹¹) in strains obtained from Brassica. The heterozygosity at the Ldh-2 locus, differing in allele composition, however, between strains from Allium and Brassica, was present in all strains, indicating that it is probably fixed. Sequence analysis of the ITS regions and the 5.8S subunit showed consistent differences between isolates from Allium and isolates from Brassica. Based on isozyme data, ITS sequence analysis and formerly published differences in restriction enzyme patterns of mitochondrial DNA, morphology and pathogenicity, it was concluded that the isolates of P. porri Foister did not represent a homogeneous species. Isolates from Brassica constitute a distinct species which is described here as P. brassicae sp. nov. It was inferred from isozyme patterns, which were in no case intermediate between the two species, that P. porri and P. brassicae do not hybridize and are reproductively isolated by barriers to gene flow.     

Induction of an antioxidant enzyme system and other oxidative stress markers associated with compatible and incompatible interactions between chickpea (Cicer arietinum L.) and Fusarium oxysporum f. sp.ciceris

To ascertain if active oxygen species play a role in fusarium wilt of chickpea caused by Fusarium oxysporum f. sp. ciceris, the degree of lipid peroxidation (malondialdehyde formation) and the activity levels of diamine oxidase (DAO), an apoplastic H₂O₂-forming oxidase, and several antioxidant enzymes, namely ascorbate peroxidase (APX), catalase (CAT), glutathione reductase (GR), guaiacol-dependent peroxidase (GPX) and superoxide dismutase (SOD), were determined spectrophotometrically in roots and stems of ‘WR315’ (resistant) and ‘JG62’ (susceptible) chickpea cultivars inoculated with the highly virulent race 5 of the pathogen. Moreover, APX, CAT, GPX and SOD were also analysed in roots and stems by gel electrophoresis and activity staining; and the protein levels of APX and SOD in roots were determined by Western blotting. In roots, infection by the pathogen increased lipid peroxidation and CAT and SOD activities, although such responses occurred earlier in the incompatible compared with the compatible interactions. APX, GPX and GR activities were also increased in infected roots, but only in the compatible interaction. In stems, infection by the pathogen increased lipid peroxidation and APX, CAT, SOD and GPX activities only in the compatible interaction, and DAO activity only in the incompatible one. In general, electrophoregrams agreed with the activity levels determined spectrophotometrically and did not reveal any differences in isoenzyme patterns between cultivars or between infected and non-infected plants. Further, Western blots revealed an increase in the root protein levels of APX in the compatible interaction and in those of SOD in both compatible and incompatible interactions. In conclusion, whereas enhanced DAO activity in stems, and earlier increases in lipid peroxidation and CAT and SOD activities in roots, can be associated with resistance to fusarium wilt in chickpea, the induction of the latter three parameters in roots and stems along with that of APX, GR (only in roots) and GPX (only in stems) activities are rather more associated with the establishment of the compatible interaction.     

Identification ofTomato Mottle Taino Begomovirus strains in Cuban potato fields

The presence of a begomovirus in potato plants with yellow mottle symptoms was determined for the first time in Cuba. The incidence of typical begomovirus-like symptoms in potato plants in some regions of Havana province (Güira de Melena, San José de las Lajas, Güines and Boyeros) during the growing seasons from 1992 to 1998 was in general low. However, in some cultivars belonging to the National Program for Potato Genetic Improvement, the incidence reached 100%. Yield losses, determined in 1992 and 1994, ranged as high as 19% to 56.33% depending on the cultivar. Characterization of the causal agent was done by light microscopy, host range (graft and mechanical transmission), DNA hybridizations, polymerase chain reaction, and restriction fragment length polymorphism analysis. Nucleotide sequence of the amplified fragments revealed the presence ofTomato mottle Taino virus. The virus was transmittedvia tubers and has been detected in mixed infections withPotato virus X and withPotato leaf roll virus. http://www.phytoparasitica.org posting Oct. 20, 2003. The first two authors contributed equally to this work.     

Indole-3-acetic acid biosynthesis in <Emphasis Type="Italic">Aciculosporium take</Emphasis>, a causal agent of witches' broom of bamboo

 Aciculosporium take (Ascomycota; Clavicipitaceae) is a causal agent of witches' broom of bamboo plants. The symptoms of this disease are believed to be induced by plant hormones, particularly auxins. Indole-3-acetic acid (IAA) was identified in cultures of this fungus in an l-tryptophan-supplemented liquid medium. IAA production was confirmed on 30 isolates of A. take from various hosts and locations at levels up to 1 mg/l. The biosynthetic pathway of IAA in A. take culture was examined by analyzing intermediate products and by feeding experiments. The results showed that the indole-3-pyruvic acid pathway (l-tryptophan → indole-3-pyruvic acid → indole acetaldehyde → IAA) was the dominant pathway in A. take. Received: June 3, 2002 / Accepted: July 25, 2002     

Evidence that a Leaf-Disk Test Allows Assessment of Isolate-Specific Resistance in Brassica oleracea Crops Against Downy Mildew (Peronospora parasitica)

A rapid resistance/susceptibility test for Peronospora parasitica (downy mildew) was established by inoculating leaf-disks of four Brassica oleracea accessions. Several conditions were tested: disk disinfection or not, agar medium with or without nutrients and with 50 or 100 ppm of benzimidazole. Using disinfected disks placed on agar (no nutrient and benzimidazole at 50 or 100 ppm), the responses of leaf-disks to four isolates were similar to those obtained using the classical cotyledon test, whereas undesired contaminations occurred in all other conditions. The possible effect of the particular leaf used for obtaining the disks was also studied. In each incompatible interaction tested, disks were resistant whatever the leaf used. In compatible interactions, susceptible phenotypes were observed on disks derived from the six lowest leaves, but disks from upper leaves were resistant. The genetic basis of resistance in a F1 hybrid broccoli was assessed, by testing six isolates on an F2 population derived from this hybrid. The cotyledon test only allows inoculation of two isolates per seedling, whereas many isolates can be tested on each plant by using leaf-disks. The segregation of the resistance to each of the six isolates was analysed: two dominant genes (tightly linked) control resistance to all isolates (one to five isolates; the other to only one isolate).     

Effect of chitin on biological control activity of Bacillus spp. and Trichoderma harzianum against root rot disease in pepper (Capsicum annuum) plants

Two bacterial isolates and one strain of Trichoderma harzianum were tested alone and in combination with chitin for efficacy in control of root rot disease caused by Phytophthora capsici and Rhizoctonia solani in pepper plants under greenhouse conditions. These bacteria (Bacillus subtilis HS93 and B. licheniformis LS674) were isolated from repeatedly washed roots of pepper plants. In in vitro assays, HS93, LS674 and T. harzianum were antagonistic against P. capsici and R. solani and produced high levels of chitinase. Seed treatment and root drenching with bacterial suspensions of HS93 with 0.5% chitin was more effective against Phytophthora and Rhizoctonia root rot than addition of the organisms without chitin. LS674 and T. harzianum reduced Rhizoctonia but not Phytophthora root rot. In two greenhouse tests, seed treatment and root drenching with HS93 amended with chitin enhanced its biocontrol activity against P. capsici but not on R. solani. The effects of LS674 and T. harzianum against R. solani were significantly enhanced when they were used as suspensions with 0.5% chitin for root drenching, but this had no effect on P. capsici. In both greenhouse experiments, the use of 0.5% chitin alone for root drenching reduced Rhizoctonia root rot. Reduction of root rot disease was accompanied by increased yield. These results show that the antagonistic activity of HS93, LS674 and T. harzianum may be stimulated by chitin resulting in significant improvements in their effectiveness against pathogens.     

Inconspicuous restraint of rice seedling growth by root-infecting fungi in soil of a rice paddy field

 Rice seedling growth, estimated by plant height and root development and discoloration, was better in pasteurized soil than in unpasteurized soil obtained from a flooded rice field. Rice seedlings also grew better in sterilized soil modified by adding roots harvested from the pasteurized soil than in soil modified by adding roots harvested from the unpasteurized soil. The results demonstrate that seedling growth in the rice field soil was inhibited by soil microorganisms, even though no typical symptoms such as seedling blight or damping-off appeared. Pythium aristosporum is suggested to be involved in the inhibition. Thus, it appears that inconspicuous restraint of rice seedling growth could occur in soils of rice paddy fields. Received: May 20, 2002 / Accepted: October 16, 2002 Acknowledgments The authors thank Dr. T. Ichitani, former professor at Osaka Prefectural University, for providing an isolate of Pythium aristosporum for comparison, and Mr. Mitsuaki Sato of Akita Prefectural College of Agriculture for technical assistance.