Hot water treatment of Japanese pear trees is effective against white root rot caused by <Emphasis Type="Italic">Rosellinia necatrix</Emphasis> Prillieux

Hot water was dripped into the rhizosphere of Japanese pear trees (Pyrus serotina Rehd. grafted on P. betulifolia Bunge.) infested with the white root rot fungus Rosellinia necatrix Prillieux, to destroy the fungus. Isolates of R. necatrix from diseased roots of Japanese pear were vulnerable to water at temperatures above 35°C, and the fungus was eradicated from the colonized substrate when water at 35°C was provided for 3 days. The time required to eradicate R. necatrix decreased exponentially with increasing temperature. Japanese pear trees tolerated a temperature of 45°C without reduction in vigor. Field experiments demonstrated the practical use of hot water drip irrigation (HWD). HWD at 50°C completely destroyed white root rot mycelia on diseased roots, and many rootlets grew after the treatment. HWD at this temperature caused no injury to the trees. HWD of diseased orchard trees was assessed in Takamori and Iida in southern Nagano, Japan. The fungus recurred in two of four trees 28 months after treatment in Takamori and in two of ten trees 16 months after treatment in Iida. The new mycelia emerged on thick roots deep within the soil. Although there is a possibility of recurrence, HWD treatment is a practical control measure for white root rot.     

Development of real-time PCR assay using TaqMan probe for detection and quantification of <Emphasis Type="Italic">Rosellinia necatrix</Emphasis> in plant and soil

We developed real-time PCR assays using TaqMan probes to detect and quantify Rosellinia necatrix, the causal agent of white root rot in many plant species. Two sets of PCR primers and TaqMan probe indicated that their detection limits could be as low as 1 fg of template DNA. Using the real-time PCR assays with the TaqMan probes, we were able to quantify R. necatrix DNA in naturally diseased roots of Japanese pear and in artificially infested soil samples. Although the new assays were inadequate for use with naturally infested soil samples, nested PCR procedures improved the detectability of the new assays.     

Identification of putative defense-related genes in Japanese pear against <Emphasis Type="Italic">Alternaria alternata</Emphasis> infection using suppression subtractive hybridization and expression analysis

The Japanese pear pathotype of Alternaria alternata, a toxin-dependent necrotrophic pathogen, causes black spot of Japanese pear by producing the host-specific AK-toxin. Pre-inoculation with nonpathogenic A. alternata or pretreatment with an elicitor prepared from A. alternata reduced disease symptoms caused by the pathogen. Salicylic acid- and jasmonic acid-dependent signaling pathways are not involved in the induced resistance to infection by the pathogen. The expression of multiple defense-related genes in Japanese pear leaves inoculated with nonpathogenic A. alternata was examined using suppression subtractive hybridization. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank database as accessions DC993229–DC993535.     

Effects of temperature,free moisture duration and inoculum concentration on infection of sweet cherry by<Emphasis Type="Italic">Pseudomonas syringae</Emphasis> pv.<Emphasis Type="Italic">syringae</Emphasis>

The effects of temperature, free moisture duration and inoculum concentration on infection caused byPseudomonas syringae pv.syringae (Pss), on sweet cherry (Prunus avium) were investigated. Epiphytic populations ofPss are an important source of inoculum for bacterial canker and it has been demonstrated that a cyclic pattern exists during the year, from undetectable during the warm and dry periods to large populations following cool and wet periods. The effects of temperature and inoculum concentration on the infection caused byPss on immature fruits and 1-yr-old twigs were significant (P<0.001). Fruit and twig infection increased linearly in proportion to the logarithm ofPss when bacterial concentrations were higher than 10³ cfu ml⁻¹ and temperatures were between 5 and 20°C. Regardless of the inoculum concentration and the free moisture duration, fruit and twig infection was either absent or low at 5°C but it increased linearly as temperature increased from 5 to 20°C. Growth ratein vitro was very slow (0.03–0.04 cfu h⁻¹) at 5°C and fast (0.21–0.23 cfu h⁻¹) at 20°C. Therefore, it is possible that multiplication of the epiphytic populations may be significantly reduced in the field with air temperatures below 5°C. A significant (P<0.001) effect of free moisture was obtained only when a low inoculum concentration (10³ cfu ml⁻¹) was used, and a significant linear response between free moisture and disease incidence was obtained only at 10°C. An apparent threshold population ofPss higher than 10³ cfu ml⁻¹ was needed to infect immature fruits and 1-yr-old twigs of sweet cherry. http://www.phytoparasitica.org posting July 10, 2002.     

Reliable detection of unevenly distributed Verticillium dahliae in diseased olive trees

Verticillium wilt caused by Verticillium dahliae is one of the most threatening diseases of olive worldwide. For pre‐planting and post‐planting control of verticillium wilt in olive trees, availability of a rapid, reliable and non‐destructive method for detection of V. dahliae is essential. For such a method, suitable and easily performed sampling and efficient processing of samples for extraction of DNA are necessary. In this study, the suitability of young twig and leaf samples of olive trees, which are easy to collect and extract DNA from, were assessed for the detection of V. dahliae in routine procedures. The lower (about 50 cm from the tip) and top parts (about 5 cm from the tip) of twigs, as well as leaves from infected olive trees were screened for V. dahliae infection and distribution using real‐time PCR. The biomass of V. dahliae detected in individual twigs was highly variable, but there was no significant difference between mean quantities of V. dahliae DNA detected in top and lower parts of twigs. Furthermore, it was demonstrated that analysis of combined samples containing DNA extracted from five twigs of an infected tree accurately detected the presence of the pathogen. Similarly, testing combined samples of 5–10 leaves enabled reliable detection of the pathogen in an infected tree. The development of this assay enables reliable detection of V. dahliae in infected olive trees that can aid in management decisions for the implementation of integrated disease management.     

Note: Comparison of three laboratory methods to evaluate the pathogenicity and virulence of tenPseudomonas syringae pv.syringae strains on apple, pear, cherry and peach trees

The pathogenicity and virulence of ten GreekPseudomonas syringae pv.syringae strains from different hosts (citrus, pear, apple, peach and cherry) were evaluated using three different laboratory methods, which produced results in good agreement. All ten strains were virulent on apple, pear, cherry and peach trees. The extent of tissue colonized varied considerably among strains and cultivars. On excised shoots and twigs of apple and pear, strains BPI 176, BPI 203, PI 2 and PI 14 were the most virulent and strains BPI 689, BPI 992, BPI 4, BPI 20, PI 18 and PI 19 were the least virulent. On excised shoots and twigs of peach and cherry, strains BPI 176, BPI 203, PI 2, PI 14, PI 18 and PI 19 were the most virulent and strains BPI 4 and BPI 20 were the least virulent. Moderate virulence was evinced by strains BPI 689 and BPI 992. These pathogenicity assays are proposed as rapid and reproducible screening systems to evaluate the susceptibility of apple, pear, cherry and peach cultivars to this bacterial pathogen.     

Levels of fire blight (<Emphasis Type="Italic">Erwinia amylovora</Emphasis>) susceptibility of native apple,pear and quince germplasm from Lake Van Basin,Turkey

Fire blight resistance of apple, pear and quince genetic resources from Lake Van Basin (eastern Turkey) was tested using Erwinia amylovora strain Ea Van. Shoot tips of 92 native accessions (48 accessions for apple, 38 accessions for pear and 6 accessions for quince) were wounded for inoculation, and artificially inoculated with pathogenic bacteria under greenhouse conditions. The levels of resistance of accessions were classified in comparison with control varieties according to the genotype susceptibility index (GSI%) scores based on the lesion length on shoots of each genotype. Fire blight resistance of accessions consisted of five classes: resistant (R), moderately resistant (MR), moderately susceptible (MS), susceptible (S) and highly susceptible (HS). GSI% scores differed significantly among accessions from each fruit species (p < 0.01). GSI values ranged from 12.4% to 64.1% for apple genotypes, from 17.2% to 55.1% for pear genotypes, and from 17.8% to 43.4% for quince genotypes. No resistant genotypes of apple, pear and quince were observed. Seven accessions of apple, two accessions of pear and one accession of quince were MR. 25 accessions of apple, 14 accessions of pear and one accession of quince were MS. These findings indicate a considerable variation in fire blight resistance and could contribute to breeding efforts regarding fire blight resistance in apple, pear and quince.     

梨和苹果腐烂病菌不同培养表型菌株的致病性分析

The pathogenicity of three strains (F-SD-8, F-BJ-2c-2 and F-HN-2a-1) of Valsa mali var. pyri causing pear canker and one strain (F-SX-A6) of V. mali var. mali causing apple canker in China were comparatively tested by wound inoculation on in vitro twigs of pear, apple and some other woody plants, and in vivo twigs of pear. Significant pathogenicity differentiation was detected in V. mali var. pyri. Generally strains F-SD-8 and F-BJ-2c-2 were highly pathogenic on pear although their culturing characteristics differed greatly. The strain F-SX-A6 was more aggressive on apple than on pear, and the strain F-HN-2a-1 showed significant lower pathogenicity on ten pear cultivars and other seven species of woody plants. Our results confirmed that two variants of V. mali had host preference and were also aggressive to crabapple, apricot, and peach besides apple and pear. Meanwhile, strains F-SD-8 and F-BJ-2c-2 could induce the formation of pycnidia on in vivo twigs of pear, which was not observed on in vivo twigs inoculated with F-HN-2a-1 and F-SX-A6.     

Molecular and serological diversity in <Emphasis Type="Italic">Apple chlorotic leaf spot virus</Emphasis> from sand pear (<Emphasis Type="Italic">Pyrus pyrifolia</Emphasis>) in China

Apple chlorotic leaf spot virus (ACLSV) isolates from sand pear (Pyrus pyrifolia) were characterized by analyzing the sequences of their coat protein (CP) genes and serological reactivity of recombinant coat proteins (rCPs). The sequences of CP genes from 22 sand pear isolates showed a high divergence, with 87.3–100% identities at the nucleotide (nt) level and 92.7–100% identities at the amino acid (aa) level. Phylogenetic analysis on the aa sequence of CP showed that the analyzed ACLSV isolates fell into different clusters and all isolates from sand pear were grouped into a large cluster (I) which was then divided into two sub-clusters (A and B). Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), western blot and enzyme-linked immunosorbent assay (ELISA) analyses demonstrated that rCPs of eight ACLSV isolates (PP13, PP15-2, PP24, PP43, PE, PP54, PP56 and ACLSV-C) from two sub-clusters had different mobility rates and serological reactivity. The rCPs of five isolates grouped into the sub-cluster A showed stronger reactivity with antibodies against rCPs of a sand pear isolate ACLSV-BD and virions of a Japanese apple isolate P-205 than that with the antibody against a Chinese apple isolate ACLSV-C. Three isolates grouped into the sub-cluster B showed stronger reactivity with the antibody against ACLSV-C. The antigenic determinants of CPs from these eight isolates and isolates ACLSV-BD and P-205 were predicted. These results contribute to a further understanding of molecular diversity of the virus and its implication in serological detection.     

Detection and identification of the phytoplasma associated with pear decline in Taiwan

Pear decline (PD) is an important phytoplasmal disease that occurs mainly in Europe and North America. In 1994, pear trees exhibiting symptoms typical of PD disease were observed in orchards of central Taiwan. The sequence of 16S rDNA and 16S–23S rDNA intergenic spacer region (ISR) of the causative agent of pear decline in Taiwan (PDTW) were amplified with polymerase chain reaction (PCR) using a DNA template prepared from the diseased leaves. Sequence analysis of 16S rDNA revealed that the PDTW agent was closely related to the phytoplasmas of the apple proliferation group that cause diseases in stone fruits, pear and apple. Consistent with the result of 16S rDNA sequence analysis, sequence analysis of the 16S–23S rDNA ISR and putative restriction site analyses of 16S rDNA and 16S–23S rDNA ISR sequences provided further support for the view that the PDTW phytoplasma causing pear decline in Taiwan may represent a new subgroup of the apple proliferation group. According to the rDNA sequence of PDTW phytoplasma, two specific PCR primer pairs, APf2/L1n and fPD1/rPDS1, were designed in this study for the detection of the etiological agent in pear trees and insect vectors. Based on the sequence analyses of the PCR-amplified fragments, two species of pear psyllas, Cacopsylla qianli and Cacopsylla chinensis, were found to carry PDTW phytoplasma.     

Black Spot of Peach Caused by <Emphasis Type="Italic">Alternaria alternata</Emphasis> (Fr.) Keissler

A new disease of peach (Prunus persica Batsch var. vulgaris Maxim.), causing brown, sunken lesions and brownish to blackish brown spots with cracks on peach fruits, was found in Okayama prefecture, Japan, in 1995. The disease was observed not only on peach fruits but also on twigs and leaves. An Alternaria sp. was consistently isolated from these diseased fruits, twigs and leaves. The isolates were pathogenic to peach fruits and leaves. Based on the morphological characteristics, the causal fungus was identified as Alternaria alternata (Fr.) Keissler. After cross-inoculation with isolates from peach, Japanese pear and apple, the isolates were found to be pathogenic only to their original host. This is the first report on a peach disease caused by a host-specific A. alternata; therefore, the common name of black spot (`Kokuhanbyo' in Japanese) was proposed. Received 25 June 1999/ Accepted in revised form 12 October 1999     

顶斑筒天牛的研究

頂斑筒天牛是梨、苹果、沙果、李、桃等的钻蛀性害虫,幼虫蛀食当年生新梢和二年生小枝,致使枯死。一年发生一代,以末龄幼虫在被害枝內越冬,4月中下旬至5月下旬变蛹,5月上中旬至6月上旬羽化。幼虫的孵化期,始于6月上中旬,終于7月中旬。根据各期习性观察,提出冬季結合整枝,剪除被害虫枝;在幼虫初孵期間,經常检查剪去虫害枯枝;选育质优产高的短枝型品种以及注意果苗检疫等一系列措施,在实施地区,已收到較好效果。     

Seed and pollen transmission of <Emphasis Type="Italic">Apple latent spherical virus</Emphasis> in apple

To examine whether Apple latent spherical virus (ALSV) has spread among apple trees in an orchard, we surveyed 21 apple trees surrounding two ALSV-infected trees for virus infection using a double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). None of the 21 trees were infected, indicating that ALSV has not spread from the infected trees to the neighboring apple trees since it was first detected in 1984. We analyzed seed embryos and seedlings derived from infected trees and detected ALSV in 10 of 223 seed embryos (4.5%) and 10 of 227 seedlings (4.4%). From these results, we conclude that ALSV is seed-transmitted at a rate of ca. 4.5% in apple. We also analyzed seed embryos and seedlings from uninfected apple trees that were hand-pollinated with pollen from infected trees. We detected ALSV in only 1 of 260 seed embryos and in none of the 227 apple seedlings. This result indicated that the seed transmission rate via infected pollen is only 0–0.38%. In situ hybridization analysis of ALSV-infected apple flower buds showed that ALSV was present inside almost all pollen grains and in all ovary and ovule tissues, including the embryo sac and inner integument.     

Enhanced detection and isolation of the walnut pathogen <Emphasis Type="Italic">Brenneria rubrifaciens</Emphasis>, causal agent of deep bark canker

Deep bark canker (DBC) of walnut is caused by the bacterium Brenneria rubrifaciens which produces the red pigment rubrifacine. This disease of English walnut trees, is characterized by deep vertical cankers which exude sap laden with B. rubrifaciens. Although DBC is not observed on young trees, it is hypothesized that B. rubrifaciens is present in host tissue years before symptom development. Therefore, a sensitive technique would be useful in detecting B. rubrifaciens in asymptomatic trees. Tn5 mutants deficient in rubrifacine production (pig ⁻) were generated and DNA sequences from pig ⁻ mutants were used to design two primer sets; GSP1F–GSP1R and GSP2F–GSP2R. A third primer pair, BR1–BR3 was designed from the 16S rRNA gene. The three primer pairs did not amplify the diagnostic bands from members of the following bacterial genera: Agrobacterium, Erwinia, Pseudomonas, Ralstonia, and Rhizobium. In addition, no amplification was observed using DNA from the following Brenneria species, alni, nigrifluens, quercina, or salicis. All three DNA primer sets detected B. rubrifaciens in spiked greenhouse soil and infiltrated walnut leaf tissue. PCR detection limits for BR, GSP1, and GSP2 primer pairs were 254, 254, and 2.54 × 10⁴ colony forming units (CFU) respectively. Real-time PCR detection limit for BR primers was 8 CFU. The differential medium, yeast extract dextrose calcium carbonate agar (YDCA) was amended with novobiocin, and bacitracin, to enhance isolation from environmental samples. The improved detection and isolation methods described here will facilitate examination of B. rubrifaciens ecology under both nursery and orchard conditions.     

Erwinia amylovora can pass through the abscission layer of fruit-bearing twigs and invade apple fruit during fruit maturation

Invasion of apple fruit by Erwinia amylovora from fruit-bearing twigs through the abscission layer at fruit maturation was examined. Erwinia amylovora (ca. 10⁵ cfu) tagged with bioluminescence genes from Vibrio fischeri was deposited in artificial wounds on fruit-bearing twigs of apple trees grown in a containment greenhouse on September 22, 27, or October 5, 2004. On October 22, 176 apples were harvested and cut horizontally in half. The upper halves were stamped on plates of selective medium, and the lower halves were flooded with iodine solution to assess maturity. All fruit were symptomless and fully mature. The pathogen was recovered from 19 (10.8%) apples. The result showed that if at least ca. 10⁵ cfu of E. amylovora are present in fruit-bearing twigs at the time of fruit maturation, the bacteria can pass through the abscission layer into the fruit, even though the mature fruit lack symptoms.     

Fireblight in Békés County (Hungary) in 1996/20021

Fireblight (Erwinia amylovora) appeared in Hungary in 1996. Most damage occurred on apple, pear, quince and medlar, and also on the ornamentals Pyracantha, Sorbus, Cotoneaster and Crataegus. In 1996–2006, an official programme for elimination of infected parts of plants started in Békés county. This mainly concerned trees in towns and villages, since there are few pome‐fruit orchards in the county. Work teams under official direction pruned back or cut down trees. In total, some 13 000 trees were pruned back and nearly 11 000 were cut down. In addition, 21 villages were subjected to special phytosanitary measures. Infection decreased considerably between 1996 and 2002, but over 90% of the inhabited areas in the county remained subject to special measures, because of the very dispersed occurrence of fireblight.     

Identification and characterization of <Emphasis Type="Italic">Apple stem grooving virus</Emphasis> causing leaf distortion on pear (<Emphasis Type="Italic">Pyrus pyrifolia</Emphasis>) in Taiwan

A putative virus-induced disease of pear (Pyrus pyrifolia var. Hengshen) showing symptoms of reduced size of foliage and leaf distortion was observed in orchards in central Taiwan in 2004. The sap of symptomatic leaf samples reacted positively to an antiserum against Apple stem grooving virus (ASGV). Two virus cultures, designated as TS1 and TS2, were isolated from symptomatic pears. Flexuous filamentous virions of ∼ 12 × 600 nm were observed in symptomatic pear leaves and purified virus preparations. Results of back inoculation of pear seedlings with TS1 revealed that ASGV was the causal agent of the disease. Sequence analyses of the cloned coat protein (CP) genes of TS1 and TS2 shared 88–92.4% nucleotide and 90.7–97.1% amino acid identities with those of other ASGV isolates available in GenBank. The polyclonal antibody generated against ASGV TS1 has been routinely used for the detection of the ASGV-infection in the imported pear scions for quarantine purpose via enzyme-linked immunosorbent assays (ELISAs). One of 1,199 samples of pear scions imported from Japan during 2005–2007 was identified as ASGV-positive and the virus was designated as AGJP-22. The CP gene amplified from this AGJP-22 shared 97.9–98.3% amino acid identities to those of the domestic isolates and they were closely related phylogenetically. To date, these data present for the first time conclusive evidence revealing that ASGV is indeed the causal agent of the pear disease displaying symptoms of reduced size of foliage and leaf distortion in Taiwan.     

Species-specific PCRs differentiate <Emphasis Type="Italic">Rosellinia necatrix</Emphasis> from <Emphasis Type="Italic">R. compacta</Emphasis> as the prevalent cause of white root rot in Japan

Rosellinia compacta, described recently, resembles R. necatrix and also causes white root rot. Here a species-specific PCR was developed for R. compacta, and the two R. necatrix-specific primer sets already available were validated in terms of species specificity. PCRs using the primer sets for R. necatrix amplified specific products exclusively from R. necatrix isolates. The R. compacta-specific primer set exclusively detected R. compacta, which appears to be a rare but widely distributed species. We conclude that R. necatrix is the major cause of the disease in Japan but that the involvement of R. compacta should be studied further.     

Growth,population dynamics,and diversity of <Emphasis Type="Italic">Fusarium equiseti</Emphasis> in ginseng fields

Fusarium equiseti is prevalent in ginseng soil, straw mulch and in ginseng root tissues and is the cause of a root surface discolouration on ginseng grown in British Columbia. Population levels of the fungus in ginseng fields ranged from 3.8 × 10³ cfu g⁻¹ soil to 1.4 × 10⁴ cfu g⁻¹ soil and were highest at 0–5 cm soil depths compared to 10–15 cm. Soil population levels were negatively correlated with S content in soil and positively correlated with Zn levels. Barley or wheat straw added to soil significantly increased population levels under laboratory conditions. Mycelial growth in culture was highest at 26–30°C and at pH 7.2–7.8. Samples of flowers and berries, and harvested seed, contained DNA of F. equiseti detected using a Fusarium-specific DNA array and the fungus was isolated from these tissues on agar medium. A high degree of genetic variation in the EF-1 alpha gene sequence was present among 52 isolates of F. equiseti which originated from ginseng fields. At least seven clades were identified. Inoculum dispersal from straw mulch used in ginseng gardens can result in seed contamination by the fungus. In addition, fungal growth near the soil surface under warm summer conditions can result in infection and crown discolouration of ginseng roots.     

Combining sub-lethal heating and on-farm wastes: effects on <Emphasis Type="BoldItalic">Fusarium oxysporum</Emphasis> f.sp. <Emphasis Type="BoldItalic">cumini</Emphasis> causing wilt of cumin

In a 2-year field study, the effects of four heating regimes established by varying temperature or duration of heating on efficiency of on-farm wastes as soil amendments in controlling Fusarium oxysporum f.sp. cumini (Foc) causing wilt on cumin (Cuminum cyminum L.), were ascertained. Significant improvement in reduction of Foc propagules was achieved with the increase in duration and amount of heat. In 2000, mild heating under shade (heat level 4), 31.8–65.9% reduction in Foc propagules was estimated in all the amendments at 0–30 cm soil depth, which improved by 75.7–86.5% in the treatment where Foc-infested soil brought from the laboratory was exposed to direct heat (heat level 3) and amendments and irrigation were applied. Foc propagules were reduced by 76.6–88.3% when infested soil was exposed continuously to dry heat for 56 days (heat level 2), improving efficiency of amendments by 0.9–13.5% compared with heat level 3. After 56 days of exposure to dry heat, elevating the temperature by mulching amended soil with a transparent polyethylene film (50 μm thick) for 20 days (heat level 1) augmented reduction by 80.2–95.5%. In the second season, combining 0.04% of onion, Verbisina encelioides or mustard oil-cake with mustard residues (0.18%) improved the reduction in Foc propagules at all heat levels even at the lower soil depth. Maximum reduction (94.9–100%) in Foc propagules at heat levels 1 to 3 was achieved when residues of Verbisina were supplemented with those of mustard. These results suggest a new approach to improve the control of Foc by combining prolonged heating with on-farm wastes such as Verbisina residues and one summer irrigation.