First Report of Fusarium Wilt of Flue-Cured Tobacco Caused by Fusarium oxysporum f. sp. batatas in Spain.

Tobacco (Nicotiana tabaccum L.) is among the most important crops in La Vera Region (Cáceres, west-central Spain) where, since 2001, flue-cured tobacco plants showing typical symptoms of a vascular disease associated to the cyst nematode (Globodera tabacum) complex have been frequently observed (2). Symptoms observed were wilting, yellowing, and drying of the leaves, usually on one side of the plant; stunted and distorted leaves with curved midribs due to the unequal growth; and brown vascular discoloration of stems and midribs. Several diseased plants were collected during the summers of 2003 and 2004, and F. oxysporum was consistently isolated from sections of necrotic midribs. To confirm the pathogenicity of F. oxysporum isolates, plants of flue-cured tobacco (cv. Ct-681) were inoculated. Since Fusarium wilt of tobacco may be caused by F. oxysporum f. sp. vasinfectum or F. oxysporum f. sp. batatas (1), plants of cotton (cv. Acala) and sweet potato (cvs. Nemagold and Nancy Hall) were also inoculated to determine the forma specialis designation. Twenty plants per cultivar and isolate were inoculated with six isolates at the two to four true-leaf stage by pouring into the substrate 200 ml of a conidial suspension (1 × 105 CFU/ml). After inoculation, plants were incubated in a growth chamber at 28°C (day) and 24°C (night) with a 16-h photoperiod. All six F. oxysporum isolates were pathogenic to tobacco and sweet potato, but there were differences among isolates in time from inoculation to appearance of first disease symptoms (7 to 15 days after inoculation in tobacco and sweet potato) and also in disease severity 30 days after inoculation (60 to 100% wilt or mortality in tobacco and 50 to 100% in sweet potato). F. oxysporum was reisolated from stems of inoculated plants. No disease symptom was observed in cotton plants 60 days after inoculation, and F. oxysporum was not reisolated from them. Results of inoculation on the differential hosts indicated that disease symptoms in tobacco were caused by F. oxysporum f. sp. batatas. To our knowledge, this is the first report of F. oxysporum f. sp. batatas causing disease in flue-cured tobacco in Spain. References: (1) G. M. Armstrong and J. K. Armstrong. Phytopathology 58:1242, 1968. (2) G. Espárrago and I. Blanco. Plant Dis. 86:1402, 2002.

First Report of the Cyst Nematode (Globodera tabacum) Complex on Flue-Cured Tobacco in Spain.

The Globodera tabacum complex infects tobacco (Nicotiana tabacum L.) fields in the United States. In August 2001, plants of flue-cured tobacco cv. K326 from a field of the La Vera Region of Spain displayed a premature wilting and yellowing of foliage, but the roots looked healthy. In the laboratory under the microscope, nematode cysts were observed on the roots. At harvest in September 2001, soil and root samples were collected to identify the nematode and to quantify the population in the soil. Identification of the nematode was based on morphological characteristics of second-stage juveniles collected from cysts and perineal patterns of cysts recovered from the roots (2). Cysts were collected from roots, and second-stage juveniles were extracted from crushed cysts. The nematode population was extracted from the soil and quantified as described by Barker (1). The nematode population was identified as Globodera tabacum. Soil density of the nematode was 5,307 cysts per liter of soil, 64,286 eggs per liter of soil, and 16,071 second-stage juveniles per liter of soil. To our knowledge, this is the first report of G. tabacum complex in Spain. References: (1) K. R. Barker. Nematode extraction and bioassays. Page 19 in: An Advanced Treatise on Meloidogyne. Vol II, Methodology. K. R. Barker, C. C. Carter, and J. N. Sasser, eds. North Carolina State University Graphics, Raleigh, 1985. (2) R. H. Mulvey and A. Morgan Golden, J. Nematol. 15:1, 1983.

Protein variability in Meloidogyne spp. (Nematoda:Meloidogynidae) revealed by two-dimensional gel electrophoresis and mass spectrometry.

Total protein variation as revealed by two-dimensional electrophoresis (2D-E) was studied in 18 isolates from populations of Meloidogyne arenaria (six isolates), Meloidogyne incognita (10 isolates), and Meloidogyne javanica (one isolate) plus an unclassified isolate. Gels (80 x 60 x 0.75 mm) were silverstained and digitized in order to compare their protein patterns. Optical density and position of protein patterns were measured using statistical cluster analysis and computer-assisted image analysis software. Only those protein stains or positions that were clearly defined (i.e., without background) were considered. The number of positions in gels ranged from 86 to 203. Each of these positions had 95 clearly expressed proteins that were present in at least two replicates for each isolate. Spot position was considered a taxonomical character with two different states: presence (1) and absence (0). Accordingly, genetic distance was estimated among isolates and species, and a phylogenetic tree was constructed following the cladistic approach based on maximum parsimony analysis. Isolates of M. arenaria--M. javanica--Meloidogyne sp. and of M. incognita formed two separate monophyletic groups. Both groups were clearly defined on the basis of two sets of protein positions that can be considered as diagnostic characters. An attempt to identify these proteins by mass spectrometry was made. Group diagnostic proteins for M. incognita and M. arenaria (and for other proteins common to all isolates) were distinguished by protonated mass signals in the MALDI fingerprinting spectrum.

Satellite DNA as a target for PCR-specific detection of the plant-parasitic nematode Meloidogyne hapla.

The polymerase chain reaction was evaluated for its ability to amplify DNA sequences specific for the root-knot nematode Meloidogyne hapla, using oligonucleotides whose sequence was deduced from the satellite DNA previously cloned in this species as primers. As expected, ladder patterns of monomers and multimers of an approximate 150-170-bp repeat were amplified from purified genomic DNA of all the M. hapla isolates studied, while no amplification was detected with the five other Meloidogyne species tested. Moreover, the satellite DNA nature of the amplification products was confirmed through Southern-blot hybridization with the previously cloned monomeric unit. In further experiments, DNA was extracted from single females, males, juveniles, or eggs according to a simple procedure, and used as a template in PCR assays. Amplification products were obtained, whose electrophoretic patterns were always very similar to those from M. hapla genomic DNA, thus demonstrating the high sensitivity of the method. This satellite DNA-based strategy can be exploited to develop species-specific primer sets for use on a routine basis as a diagnostic tool for unambiguous nematode identification procedures.