Molecular diagnostic procedures for production of pathogen-free propagation material

Production of disease-free propagation material is a major means of controlling most bacterial diseases of plants, particularly when neither resistant clones nor effective chemical treatments are available. For this purpose sensitive, specific and rapid detection methods are required. The advent of molecular biology and, in particular, the polymerase chain reaction (PCR) has opened new ways for the characterization and identification of plant pathogens and the development of disease-management strategies. PCR-based detection methods rely on the development of primers for the specific detection of the pathogen. The use of pathogenicity genes as targets for primer design is the preferred procedure for obtaining specific primers but other procedures may also be useful for this purpose. In the present review we describe four examples of procedures for detecting four important bacterial pathogens in Israel: Erwinia herbicola pv gypsophilae in gypsophila, Xanthomonas campestris pv pelargonii in geranium, Agrobacterium tumefaciens in asters and roses, and Xanthomonas campestris pv campestris in crucifers. Procedures for constructing specific PCR primers for each bacterium are illustrated and discussed as well as the combination of PCR with other methods.     

A general method for the analysis of genetic variation in complete and incomplete diallels and North Carolina II designs. Part I: Procedures and general formulas for the random model

Summary In two papers formulas are given which can be used in analysing genetic variation in all types of connected diallel cross designs: complete and incomplete, balanced and unbalanced as well as North Carolina II designs.This first paper gives explicit expressions for the coefficients of variance components in the random model analysis for the general diallel cross.Computations are illustrated with simple balanced examples. For the same examples a fixed model analysis, introducing reduced parameters has been worked out for the sake of completeness. The details of the fixed model analysis will be presented in the second paper.     

Differential photosynthetic and survival responses to soil drought in two evergreen Nothofagus species

We asked if differences in distribution between Nothofagus nitida and N. dombeyi were associated with differences in drought tolerance. Survival, gas exchange and chlorophyll fluorescence were measured on seedlings subjected to a gradual drought. At a predawn leaf water potential (Ψ_m) of ?2.7 MPa, survival of N. nitida was 50%, compared to 100% in N. dombeyi. Under well-watered conditions, the two species displayed similar stomatal conductance (g _w ) and transpiration (E), but net photosynthesis (A) and instantaneous water-use efficiency (WUE _i ) were slightly higher in N. nitida. A, E and g _w declined in N. nitida along the gradual drought but increased in N. dombeyi at a Ψ_m between ?1.5 and ?2.5 MPa, and declined then drastically at a Ψ_m below < ?2.5 MPa. As N. dombeyi was able to maintain A at higher levels despite declining g _w , this species displayed significantly increased WUE _i at Ψ_m below ?2.5 MPa. Photochemical efficiency of PSII in the light (ΔF/Fmr) and photochemical quenching (qP) were always lower in N. nitida and along with the photochemical efficiency in the dark (Fv/Fm) they declined in both species. Non-photochemical quenching (NPQ) increased slowly in N. dombeyi along with the gradual drought, whilst it decreased in N. nitida. These results show that differences in drought tolerance are in agreement with sorting of Nothofagus species along moisture gradients in south-central Chile.     

Growth analyses of tomato genotypes grown under low night temperatures and low light intensity

Summary In three experiments the growth of 16 genotypes of tomato was examined. The plants were raised in growth rooms at a day temperature of 19°C and night temperatures of 14, 10 or 6°C respectively under a light intensity of 24 Wm^-2 visible radiation and a daylength of 8 hours. The results were analysed by a multivariate analysis of variance.Relative growth rate (RGR), leaf area ratio (LAR), specific leaf area (SLA) and leaf weight ratio (LWR) decreased with time. The change in net assimilation rate (NAR) was small. The decrease in RGR was mainly due to the decrease in LAR.RGR, LAR and SLA were lower and LWR slightly higher at lower night temperatures. NAR was hardly affected. The lower RGR at lower night temperatures was mainly due to a lower SLA.Significant differences between genotypes were found in total dry weight, NAR, LAR, SLA and LWR, but hardly in RGR. Significant genotype × temperature interactions occurred.LAR and SLA were positively, NAR and LAR, and NAR and SLA negatively correlated.     

Use of a diagnostic medium for<Emphasis Type="Italic">in situ</Emphasis> determination of the response of<Emphasis Type="Italic">Erwinia amylovora</Emphasis> strains to bactericides

Erwinia amylovora, the causal agent of fire blight, is managed by application of bactericides to protect fruit tree blossoms from infection. Monitoring the response ofE. amylovora strains to bactericides is crucial for adequate disease management. The coliform agar medium produced by Merck was recently reported as an effective tool for rapid diagnosis ofE. amylovora (RD-medium). The objective of the present study was to examine the possibility of using the RD-medium forin situ determination of the response ofE. amylovora strains to oxolinic acid and streptomycin. The phenotypic response of 48E. amylovora strains isolated in 2002 to both bactericides was determined with the RD-medium and, for comparison, by a routine laboratory test. The results of 45 samples (93.7%) were in agreement with the findings of the routine laboratory test. Aχ ² test rejected the null hypothesis that the phenotypic characteristics as determined by the two respective methods differed significantly (P=0.389). Thein situ test was implemented on a national scale in 2003 and the results were in agreement with those obtained in laboratory tests, which suggests that this medium can be usedin situ for monitoring the appearance of resistance inE. amylovora populations. http://www.phytoparasitica.org posting Feb. 11, 2004.     

Characterization of a Clavibacter michiganensis subsp. michiganensis population in Israel

Clavibacter michiganensis subsp. michiganensis (Cmm) strains, collected during the last decade from different locations in Israel, were analyzed by macrorestriction pulsed-field gel electrophoresis (PFGE). Fifty-eight strains from Israel and 18 from other sources were differentiated into 11 haplotypes with either VspI or DraI restriction enzymes. The strains from Israel formed four distinct groups among which groups A (16 strains) and B (32 strains) constituted the major clusters. These two groups originated from the Besor region, which is the main area for growing tomatoes under cover. Rep-PCR, with either ERIC or BOX primers, confirmed results obtained by PFGE. PCR with primers based on three genes – ppaA, chpC and tomA – that spanned the pathogenicity island of the reference strain NCPPB382, produced the expected products with the tested pathogenic strains. Plasmid analysis of representative strains revealed different profiles of one or two plasmids. However all the strains, including five non-pathogenic ones, reacted positively in PCR with primers based on celA gene, which resides on the plasmid pCM1 of NCPPB382. Southern hybridization of total DNA with a 3.2-kb BglII-fragment of pCM1 containing the celA gene was positive when carried out with 31 strains, but the size of the reacting band was not always the same as that of pCM1, suggesting that the plasmids carrying celA may differ in size. Comparison between the colonization rates of strain Cmm42 (group A) and of Cmm32 (group B) did not show any significant differences. The high diversity of the Cmm strains, on the one hand, and the presence of two persistent groups in the Besor region, on the other hand, suggests that the primary inoculum originated each year from residual plants in the soil rather than from infested seeds, in spite of extensive control measures taken by the growers in this area.     

Distribution of streptomycin-resistant strainsof Erwinia amylovora in Israel and occurrence of blossom blight in the autumn

Following failure in control of fire blight with streptomycin, the distribution of streptomycin-resistant strains ofErwinia amylovora in Israel was surveyed. During 1994–1997 109 pear, apple, loquat and quince orchards were monitored. Streptomycin-resistant strains ofE. amylovora were recovered from flowers and from infected branches collected at 18 locations in the Sharon, Galilee and Golan Heights regions. In the Sharon region all the isolated strains ofE. amylovora were streptomycin-resistant, whereas in the Galilee and Golan Heights, resistant as well as sensitiveE. amylovora strains were recovered at different locations. In the southern coastal plain no resistance could be detected. Streptomycin-resistant strains ofE. amylovora did not hybridize with the DNA probe SMP3, and resistance could not be transferred by mating to a sensitive strain, suggesting that streptomycin resistance in Israel is not plasmid-mediated. Fire blight symptoms were observed, for the first time, on pear blossoms during the autumn of 1994. A high population of 2x 10⁶-6x 10⁷ CFU/flower in the autumn of 1995 and of 1996 was correlated with the appearance of blossom blight symptoms.     

Genetic analysis of cut flower longevity in Gerbera

Summary The vase life duration and stem stiffness of 12 parent clones and 77 progenies of Gerbera jamesonii were studied both in summer and in winter. In winter, vase life was shorter and the stems were weak. Few exceptions from this general pattern were detected. The average vase life duration ranged from 16.0 days for the best to 8.9 days for the worst progeny. Individual plants were selected with a vase life of 20 or more days.The curvature of the stem, measured after 24 hrs dry storage, is associated with the folding of the stem in the vase. Selection for low curvature may decrease the incidence of folding and with it the variation in vase life of Gerbera.     

Genetic analysis of flower and lateral shoot production in Gerbera

Summary An incomplete diallel cross with selfs and reciprocals was made with twelve cultivars of Gerbera jamesonii. Very significant differences occurred between GCA's of the parents for cut flower yield, earliness and number of lateral shoots. The selfs were mainly responsible for the significant SCA's.A positive genetic correlation occurs between the number of lateral shoots at anthesis of the first flower and total flower production. The phenotypic performance of parents (measured on cuttings) was poorly related to their breeding value (measured on seedlings). It is suggested that this is due to physiological differences between cuttings and seedlings.