Effect of Prohexadione-Calcium Dose Level on Shoot Growth and Fire Blight in Young Apple Trees.

Prohexadione-calcium suppresses both shoot growth and fire blight in apple. In young apple orchards, there are conflicting requirements to control fire blight and allow sufficient tree growth for tree establishment. Application of prohexadione-calcium to various cultivars of orchard-grown apple trees ranging in age from newly planted to fifth-leaf trees indicated that fewer high-dose (125 or 250 mg ·liter-1) applications of prohexadione-calcium provided a better balance between fire blight control and growth in young orchards than multiple low-dose (30 or 63 mg·liter-1) applications. The response of early-season shoot growth to prohexadione-calcium treatment dose was linear. However, trees that received high doses of prohexadione-calcium tended to grow more in the latter part of the season, resulting in little or no difference in total seasonal growth between trees that received a few high or multiple low doses of prohexadione-calcium. Enhancement of fire blight resistance by prohexadione-calcium was correlated with shoot growth suppression at the time of inoculation, and the resistance response to prohexadione-calcium treatment dose was linear. Fire blight management strategies that use prohexadione-calcium in young apple orchards are discussed.

Resistance of Geneva and Other Apple Rootstocks to Erwinia amylovora.

When vigorously growing shoots of 49 different apple rootstocks grown in a greenhouse were inoculated with different strains of Erwinia amylovora, Budagovsky 9 (B.9), Ottawa 3, Malling 9, and Malling 26 were the most fire blight susceptible rootstocks and Geneva 11, Geneva 65, Geneva 16, Geneva 30, Pillnitzer Au51-11, Malling 7, and several breeding selections were the most resistant. Significant strain-rootstock interactions were observed in the amount of fire blight that resulted from inoculation. Field-grown fruiting 'Royal Gala' trees on Geneva 16 and Geneva 30 rootstocks were highly resistant to rootstock infection (no tree mortality) when trees sustained severe blossom infection with E. amylovora, compared with Malling 9 and Malling 26 rootstock clones, which were highly susceptible to infection (36 to 100% tree mortality). In contrast to potted own-rooted B.9 plants inoculated in a greenhouse, B.9 rootstocks of orchard trees appeared resistant to rootstock infection (0% tree mortality). Orchard trees on Geneva 11 were moderately resistant to rootstock infection (25% tree mortality). There was general agreement in the evaluation of resistance under orchard conditions when rootstock resistance was evaluated in relation to controlled blossom inoculation or to natural blossom infection.

Synergistic activity of endochitinase and exochitinase from Trichoderma atroviride (T. harzianum) against the pathogenic fungus (Venturia inaequalis) in transgenic apple plants.

Genes from the biocontrol fungus Trichoderma atroviride encoding the antifungal proteins endochitinase or exochitinase (N-acetyl-beta-D-hexosaminidase) were inserted into 'Marshall McIntosh' apple singly and in combination. The genes were driven by a modified CaMV35S promoter. The resulting plants were screened for resistance to Venturia inaequalis, the causal agent of apple scab, and for effects of enzyme expression on growth. Disease resistance was correlated with the level of expression of either enzyme when expressed alone but exochitinase was less effective than endochitinase. The level of expression of endochitinase was negatively correlated with plant growth while exochitinase had no consistent effect on this character. Plants expressing both enzymes simultaneously were more resistant than plants expressing either single enzyme at the same level; analyses indicated that the two enzymes acted synergistically to reduce disease. Selected lines, especially one expressing low levels of endochitinase activity and moderate levels of exochitinase activity, were highly resistant in growth chamber trials and had negligible reduction in vigor relative to control plants. We believe that this is the first report of resistance in plants induced by expression of an N-acetylhexosaminidase and is the first report of in planta synergy between an exochitinase and an endochitinase.

Expression of Endochitinase from Trichoderma harzianum in Transgenic Apple Increases Resistance to Apple Scab and Reduces Vigor.

ABSTRACT The goal of this research was to improve scab resistance of apple by transformation with genes encoding chitinolytic enzymes from the bio-control organism Trichoderma harzianum. The endochitinase gene, as cDNA and genomic clones, was transferred into apple cv. Marshall McIntosh by Agrobacterium-transformation. A total of 15 lines were identified as transgenic by NPTII enzyme-linked immunosorbent assay and polymerase chain reaction and confirmed by Southern analysis. Substantial differences in endochitinase activity were detected among different lines by enzymatic assay and western analysis. Eight lines propagated as grafted and own-rooted plants were inoculated with Venturia inaequalis. Six of these transgenic lines expressing endochitinase were more resistant than nontransformed cv. Marshall McIntosh. Disease severity compared with cv. Marshall McIntosh was reduced by 0 to 99.7% (number of lesions), 0 to 90% (percentage of leaf area infected), and 1 to 56% (conidia recovered) in the transgenic lines tested. Endochitinase also had negative effects on the growth of both inoculated and uninoculated plants. There was a significant negative correlation between the level of endochitinase production and both the amount of disease and plant growth.

Internal Movement of Erwinia amylovora Through Symptomless Apple Scion Tissues into the Rootstock.

Shoot tips of potted Empire and Golden Delicious trees on the susceptible dwarfing rootstock M.26 in the greenhouse were injected with inoculum containing 5 × 109 CFU/ml Erwinia amylovora. At intervals after inoculation, trees were sampled at increments between the shoot tip and the roots by excising stem segments. Segments were ground in phosphate buffer and assayed for the presence of E. amylovora by plating on semi-selective medium and by a polymerase chain reaction (PCR)-based detection method. Eleven days after inoculation, E. amylovora was detected by PCR in symptomless scion tissue >50 cm below the shoot-tip in Empire and Golden Delicious, and in 2-year-old tissue in Golden Delicious. By 21 days, E. amylovora was detected in the M.26 rootstock of Empire trees, and by 41 days in the M.26 rootstock of Golden Delicious. In a similar experiment the following year, Empire trees on M.26 rootstock were inoculated with E. amylovora at early (16 May), mid- (11 June), and late (6 July) phenophase of shoots. Three weeks after inoculation, E. amylovora was detected by PCR from M.26 rootstocks of five of six plants inoculated at the late phenophase, compared to zero of six plants inoculated at the early or mid-phenophase. Late-season fire blight infections of the scion may be particularly hazardous for the health of the rootstock.

Characterization of Erwinia amylovora strains using random amplified polymorphic DNA fragments (RAPDs).

The genetic diversity among 16 strains of Erwinia amylovora, chosen to represent different host plant origins and geographical regions, was investigated by RAPD analysis. One strain of Erwinia herbicola and one of Agrobacterium vitis were used as outgroups. Ninety-eight different RAPD fragments were produced by polymerase chain reaction amplification with six different 10-mer primers. RAPD banding profiles were found that enabled the Erw. amylovora strains to be distinguished from one another. Cluster analysis based on the number of RAPD fragments shared between strains showed that strains of Erw. amylovora isolated from subfamily Pomoideae formed a single group, whereas two strains from Rubus (subfamily Rosoideae) formed a second group. Two strains isolated from Asian pear on Hokkaido, Japan, formed a third group. Sets of RAPD fragments were identified that enabled each of the two host-range groups and one geographical region (Hokkaido) of Erw. amylovora strains to be unambiguously distinguished from one another and from the outgroups. This study shows that strains of Erw. amylovora exhibit genetic diversity detectable by RAPD analysis, and that molecular and statistical analysis of RAPD fragments can be used both to distinguish between strains and to determine relatedness between them.

Homologous Streptomycin Resistance Gene Present among Diverse Gram-Negative Bacteria in New York State Apple Orchards.

The streptomycin resistance gene of Pseudomonas syringae pv. papulans Psp36 was cloned into Escherichia coli and used to develop a 500-bp DNA probe that is specific for streptomycin resistance in P. syringae pv. papulans. The probe is a portion of a 1-kb region shared by three different DNA clones of the resistance gene. In Southern hybridizations, the probe hybridized only with DNA isolated from streptomycin-resistant strains of P. syringae pv. papulans and not with the DNA of streptomycin-sensitive strains. Transposon insertions within the region of DNA shared by the three clones resulted in loss of resistance to streptomycin. Colony hybridization of bacteria isolated from apple leaves and orchard soil indicated that 39% of 398 streptomycin-resistant bacteria contained DNA that hybridized to the probe. These included all strains of P. syringae pv. papulans and some other fluorescent pseudomonads and nonfluorescent gram-negative bacteria, but none of the gram-positive bacteria. The same-size restriction fragments hybridized to the probe in P. syringae pv. papulans. Restriction fragment length polymorphism of this region was occasionally observed in strains of other taxonomic groups of bacteria. In bacteria other than P. syringae pv. papulans, the streptomycin resistance probe hybridized to different-sized plasmids and no relationship between plasmid size and taxonomic group or between plasmid size and orchard type, soil association, or leaf association could be detected.

Use of ti plasmid DNA probes for determining tumorigenicity of agrobacterium strains.

Probes consisting of T-DNA genes from the Ti plasmid of Agrobacterium tumefaciens were used for determining tumorigenicity of strains. Two P-labeled probes hybridized with 28 of 28 tumorigenic strains of the pathogen but not with 20 of 22 nontumorigenic strains. One probe, pTHE17, consists of all but the far left portion of the T-DNA of strain C58. Probe SmaI7 consists of SmaI fragment 7 of pTiC58, including onc genes 1, 4, and 6a and most of 2. Another probe, pAL4044, consisting of the vir region of strain Ach-5, hybridized with several nontumorigenic as well as tumorigenic strains. Colony hybridizations were done with 28 tumorigenic and 22 nontumorigenic Agrobacterium strains. About 10 CFU of the different tumorigenic strains were detectable with this method. Southern analyses confirmed the presence or absence of Ti plasmids in strains for which tumorigenicity was questioned. Colony hybridization with the T-DNA probes provides a rapid and sensitive means for determining the tumorigenic nature of Agrobacterium strains.