Infection and colonization of strawberry by <Emphasis Type="Italic">Gnomonia fragariae</Emphasis> strain expressing green fluorescent protein

Gnomonia fragariae is a poorly studied ascomycete, which was recently demonstrated to be a cause of severe root rot and petiole blight of strawberry. The pathogen was genetically transformed with the GFP as a vital marker and hygromycin resistance gene. Several stable transformants were obtained, which did not differ in their phenotype from the wild type isolate. Using one of the GFP-tagged isolates the infection process and colonization of roots and petioles of host plant by the pathogen were studied. Fluorescence microscopy examinations of the inoculated plants at different time points showed that plant infection occurs 24 h after inoculation and intensively continues during first 3 days. The specific penetration sites on epidermal cells and preferences in colonization for certain root and petiole tissues were observed. The pathogen intensively colonized and destroyed cortex of roots and petioles and spread rapidly longitudinally within intercellular spaces. The petioles were colonized by the hyphae, which grew mostly in the intracellular spaces of the cortical cells while in the roots the intracellular growth of hyphae occurred only in the later stages of infection. The fungus was also capable to infect the vascular tissues of petioles although these were not the primary tissues colonized by the pathogen. The mature ascomata were formed on the infected petiole bases several weeks after the inoculation. This study presents a genetic transformation method for Gnomonia fragariae and it demonstrates details on infection process and colonization of root, crown and petiole tissues of strawberry by the pathogen.     

Effect of Phenylpyrrole-resistance Mutations on Ecological Fitness of <Emphasis Type="Italic">Botrytis cinerea</Emphasis> and their Genetical Basis in <Emphasis Type="Italic">Ustilago maydis</Emphasis>

Mutants of Botrytis cinerea and Ustilago maydis highly resistant to fludioxonil were isolated at a high frequency, after nitrosoguanidine or UV mutagenesis, respectively, and selection on media containing fludioxonil. Tests on the response of mutant strains to high osmotic pressure resulted in the identification of two fludioxonil-resistant phenotypes (FLD_osm/s and FLD_osm/r), regarding the sensitivity to high osmolarity. Approximately 95% of fludioxonil-resistant mutants were found to be more sensitive to high osmotic pressure than the wild-type parent strains. Genetic analysis of phenylpyrrole-resistance in the phytopathogenic basidiomycete U. maydis, showed that fludioxonil-resistance was coded by three unlinked chromosomal loci (U/fld-1, U/fld-2 and U/fld-3), from which only the U/fld-1 mutation coded an osmotic sensitivity similar to that of the wild-types. Cross-resistance studies with fungicides from other chemical groups showed that the mutations for resistance to phenylpyrroles affect the sensitivity of mutant strains to the aromatic hydrocarbon and dicarboximide fungicides, but not to the benzimidazoles, anilinopyrimidines, phenylpyridinamines, hydroxyanilides or the sterol biosynthesis inhibiting fungicides. A study of fitness parameters in the wild-type and fludioxonil-resistant mutants of B. cinerea, showed that all osmotic sensitive (B/FLD_osm/s) isolates had significant reductions in the characteristics determining saprophytic fitness such as mycelial growth, sporulation, conidial germination and sclerotial production. Contrary to that, with the exception of mycelial growth, the fitness parameters were unaffected or only slightly affected in most of the osmotic resistant (B/FLD_osm/r) isolates. Tests on cucumber seedlings showed that the osmotic-sensitive strains were significantly less pathogenic compared with the wild-type and B/FLD_osm/r strains of B. cinerea. Preventative applications of the commercial products Saphire 50 WP (fludioxonil) and Rovral 50 WP (iprodione) were effective against lesion development on cotyledons by the wild-type and the mutant strains of B. cinerea that were resistant to the anilinopyrimidine cyprodinil (B/CPL-27) and to the hydroxyanilide fenhexamid (B/FNH-21), but ineffective, even at high concentrations, against disease caused by the fludioxonil-resistant isolates (B/FLD) and a mutant strain resistant to the dicarboximide iprodione (B/IPR-1). Experiments on the stability of the fludioxonil-resistant phenotype showed a reduction of resistance, mainly in osmotic-sensitive isolates, when the mutants were grown on inhibitor-free medium. A rapid recovery of the high resistance was observed after mutants were returned to the selection medium. Studies on the competitive ability of mutant isolates against the wild-type parent strain of B. cinerea, by applications of a mixed conidial population, showed that, in vitro, all mutants were less competitive than the wild-type strain. However, the competitive ability of osmotic-resistant mutants was higher than the osmotic-sensitive ones. Furthermore, competition tests, in planta, showed a significant reduction of the frequency of both phenylpyrrole-resistant phenotypes, with a respective increase in the population of the wild-type strain of the pathogen.     

Visualisation of <Emphasis Type="Italic">hrp</Emphasis> gene expression in <Emphasis Type="Italic">Xanthomonas euvesicatoria</Emphasis> in the tomato phyllosphere

The plasmid pUFZ75 conferred constitutive GFP expression on the bacterial pathogen Xanthomonas euvesicatoria (syn. X. campestris pv. vesicatoria). Colonisation of the tomato phyllosphere and invasion of tomato leaves by X. euvesicatoria was examined using both fluorescence and confocal laser scanning microscopy. Xanthomonas euvesicatoria established a limited population on the tomato leaf surface, primarily occupying the depressions between epidermal cells and around the stomata, prior to invasion of the leaf via the stomata and subsequent growth within the substomatal chamber and the leaf apoplast. Additionally, hrp-gfp fusions were used to report on the temporal and spatial expression of hrp genes during epiphytic colonisation and invasion. Xanthomonas euvesicatoria cells carrying hrpG- and hrpX-gfp reporter constructs were not fluorescent in vitro on non-hrp-inducing LB agar but did exhibit a low level of fluorescence on the leaf surface within 24 h of inoculation, particularly in the vicinity of stomata. Cells carrying hrpG- and hrpX-gfp fusions exhibited high levels of fluorescence 72 h after inoculation in the substomatal chamber and the leaf apoplast. Cells carrying the hrpF-gfp fusion were slightly fluorescent on LB agar and showed no further increase in fluorescence on the leaf surface by 24 h after inoculation, but did show a significant increase in fluorescence 72 h after inoculation in the substomatal chamber and apoplast. The apparent low-level induction of the regulators hrpG and hrpX on the tomato leaf surface may suggest that some of the genes of the X. euvesicatoria HrpG/HrpX regulon are up- or down-regulated prior to invasion of the stomata while still on the leaf surface.     

Functional analysis of the melanin biosynthesis genes <Emphasis Type="Italic">ALM1</Emphasis> and <Emphasis Type="Italic">BRM2</Emphasis>-<Emphasis Type="Italic">1</Emphasis> in the tomato pathotype of <Emphasis Type="Italic">Alternaria alternata</Emphasis>

The tomato pathotype of Alternaria alternata (A. arborescens) produces the dark brown to black pigment melanin, which accumulates in the cell walls of hyphae and conidia. Melanin has been implicated as a pathogenicity factor in some phytopathogenic fungi. Here, two genes of the tomato pathotype for melanin biosynthesis, ALM1 and BRM2-1, which encode a polyketide synthetase and a 1,3,8-trihydroxynaphthalene (THN) reductase, respectively, have been cloned and disrupted in the pathogen. The gene-disrupted mutants, alm1 and brm2-1, had albino and brown phenotypes, respectively. The wild-type and the mutants caused the same necrotic lesions on the leaves after inoculation with spores. These results suggest that melanin is unlikely to play a direct role in pathogenicity in the tomato pathotype A. alternata. Scanning electron microscopy revealed that the conidia of both mutants have much smoother surfaces in comparison to the wild-type. The conidia of those mutants were more sensitive to UV light than those of the wild-type, demonstrating that melanin confers UV tolerance.     

<Emphasis Type="Italic">Botrytis cinerea</Emphasis> induces senescence and is inhibited by autoregulated expression of the <Emphasis Type="Italic">IPT</Emphasis> gene

Botrytis cinerea is a non-specific, necrotrophic pathogen that attacks many plant species, including Arabidopsis and tomato. Since senescing leaves are particularly susceptible to infection by B. cinerea, we hypothesized that the fungus might induce senescence as part of its mode of action and that delaying leaf senescence might reduce the severity of B. cinerea infections. To examine these hypotheses, we followed the expression of Arabidopsis SAG12, a senescence-specific gene, upon infection with B. cinerea. Expression of SAG12 is induced by B. cinerea infection, indicating that B. cinerea induces senescence. The promoter of SAG12, as well as that of a second Arabidopsis senescence-associated gene, SAG13, whose expression is not specific to senescence, were previously analyzed in tomato plants and were found to be expressed in a similar manner in the two species. These promoters were previously used in tomato plants to drive the expression of isopentenyl transferase (IPT) from Agrobacterium to suppress leaf senescence (Swartzberg et al. in Plant Biology 8:579–586, 2006). In this study, we examined the expression of these promoters following infection of tomato plants with B. cinerea. Both promoters exhibit high expression levels upon B. cinerea infection of non-senescing leaves of tomato plants, supporting our conclusion that B. cinerea induces senescence as part of its mode of action. In contrast to B. cinerea, Trichoderma harzianum T39, a saprophytic fungus that is used as a biocontrol agent against B. cinerea, induces expression of SAG13 only. Expression of IPT, under the control of the SAG12 and SAG13 promoters in response to infection with B. cinerea resulted in suppression of B. cinerea-induced disease symptoms, substantiating our prediction that delaying leaf senescence might reduce susceptibility to B. cinerea. Contribution from the Agriculture Research Organization, The Volcani Center, Bet Dagan, Israel, No. 127/2006 series.     

Visualization of <Emphasis Type="Italic">Ralstonia solanacearum</Emphasis> cells during biocontrol of bacterial wilt disease in tomato with <Emphasis Type="Italic">Pythium oligandrum</Emphasis>

The biocontrol agent Pythium oligandrum (PO) can suppress bacterial wilt caused by Ralstonia solanacearum (RS) in tomato. To understand the primary biocontrol mechanisms of bacterial wilt by PO, we pretreated tomato plants with sterile distilled water or preinoculated them with PO, followed by inoculation with RS, then observed PO and RS in fixed sections of tomato tissues using a confocal laser-scanning microscope and fluorescence labeling until 14 days after the inoculation with RS. Horizontal and vertical movement of RS bacteria was frequently observed in the xylem vessels of roots and stems of tomato plants (cv. Micro-Tom) that had not been inoculated with PO. In plants that were preinoculated with PO, the movement of RS was suppressed, and bacteria appeared to be restricted to the pit of vessels, a reaction similar to that observed in resistant rootstocks. PO colonization was mainly observed at the surfaces of taproots, the junctions between taproots and lateral roots, and the middle sections of the lateral roots. PO was not observed near wound sites or root tips where RS tended to colonize. However, RS colonization was significantly repressed at these sites in PO preinoculated plants. These observations suggest that the induction of plant defense reactions is the main mechanism for the control of tomato bacterial wilt by PO, not direct competition for infection sites.     

Resistance of <Emphasis Type="Italic">Solanum</Emphasis> species to <Emphasis Type="Italic">Cucumber mosaic virus</Emphasis> subgroup IA and its vector <Emphasis Type="Italic">Myzus persicae</Emphasis>

Sixty-nine tomato genotypes representing nine Solanum species were evaluated for resistance to Cucumber mosaic virus (CMV) subgroup IA and its aphid vector Myzus persicae. Resistance was assessed by visual scoring of symptoms in the field under natural conditions, and in the greenhouse by artificial inoculations through aphid M. persicae and mechanical transmissions in the year 2007 and 2009. Considerable variation in responses was observed among the evaluation methods used. Field evaluations were found liable to errors as different levels were observed for the same genotypes in the different years, however mechanical inoculation was found to be the most useful in identifying CMV subgroup IA resistance, in contrast aphid transmission was most useful in identifying insect transmission resistance. All genotypes observed as highly resistant to CMV subgroup IA in the field or through vector transmission became systemically infected through mechanical inoculations. Using mechanical inoculation, six genotypes (TMS-1 of S. lycopersicum, LA1963 and L06049 of S. chilense, LA1353, L06145 and L06223 of S. habrochaites) were found resistant and another six (L06188 and L06238 of S. neorickii, L06219 of S. habrochaites, L05763, L05776 and L06240 of S. pennellii) were found tolerant showing mild symptoms with severity index (SI) ranging 1-2 and with delayed disease development after a latent period (LP) of 18–30 days. However, these genotypes were found to be resistant to highly resistant in the field and through inoculation by M. persicae; and they also supported low population levels of M. persicae except TMS-1. Another nine genotypes (LA2184 of S. pimpinellifolium L., LA2727 of S. neorickii, LA0111, L06221, L06127 and L06231 of S. peruvianum L., LA1306, L06057 and L06208 of S. chmielewskii) showing a susceptible response after mechanical inoculation were highly resistant, resistant and tolerant after M. persicae transmission. The resistant genotypes, identified in the present study can be exploited in the breeding programmes aimed at developing tomato varieties resistant to CMV subgroup IA and broadening the genetic base of CMV-resistant germplasm. The differences observed between mechanical and aphid transmission suggests that one should consider both evaluation methods for tomato germplasm screening against CMV subgroup IA.     

Effects of some fungicides on <Emphasis Type="Italic">Isaria farinosa</Emphasis>, and <Emphasis Type="Italic">in vitro</Emphasis> growth and infection rate on <Emphasis Type="Italic">Planococcus citri</Emphasis>

The effects of some fungicides used against citrus diseases, on mycelial growth and conidial germination of Isaria farinosa (Holmsk.) Fries [Sordariomycetes: Hypocreales] and also on the pathogenicity of the fungus on citrus mealybug, Planococcus citri (Risso), were determined. Systemic fungicides such as tebuconazole, penconazole and nuarimol were the most effective as regards both conidial germination and mycelial growth. Protective fungicides such as captan, chlorothalonil, mancozeb and propineb inhibited conidial germination at between 1 and 5 μg ml⁻¹ concentration, but captan, chlorothalonil and propineb did not inhibit the mycelial growth at 5,000 μg ml⁻¹. Mancozeb inhibited mycelial growth between 2,500 and 5,000 μg ml⁻¹. Sulphur and copper oxychloride did not inhibit the fungus even at very high concentrations. Sulphur, copper oxychloride, fosetyl-al, chlorothalonil and carbendazim did not decrease the mortality percentage caused by I. farinosa. Tebuconazole, penconazole and mancozeb were the most effective and respectively reduced the mortality from 83% to 33%, 28% and 30% in the ovisacs, from 81% to 29%, 27% and 29% in the 1st instar larvae, and from 84% to 34% in the adult females.     

Induction of phytoalexins in adzuki bean after inoculation with <Emphasis Type="Italic">Phytophthora vignae</Emphasis> f. sp. <Emphasis Type="Italic">adzukicola</Emphasis>

To identify phytoalexins of adzuki bean elicited in response to attempted infection of Phytophthora vignae f. sp. adzukicola, we isolated compounds from adzuki bean and evaluated their antifungal activity. Seven flavonoids (daidzein, genistein, 2′-hydroxygenistein, coumestrol, dalbergioidin, kievitone, and phaseol) were identified from epicotyls wound-inoculated with a mycelial suspension of an avirulent race of P. vignae f. sp. adzukicola. Of those compounds, kievitone and dalbergioidin accumulated to higher levels in incompatible interactions compared to compatible interactions 48 h after inoculation. Kievitone strongly inhibited the germination of encysted zoospores, and dalbergioidin were slightly suppressive. From these results, we concluded that kievitone and dalbergioidin are phytoalexins in adzuki bean.     

Blue mold of tomato caused by <Emphasis Type="Italic">Penicillium oxalicum</Emphasis> in Japan

In 2006, stem rot and blue-green crusty lesions were found on the stems of tomato plants in Chiba Prefecture, Japan. Penicillium oxalicum was isolated repeatedly from the diseased plants. The causal fungus reproduced natural symptoms after artificial inoculation of tomato plants and was re-isolated from symptomatic plant tissue. P. oxalicum is a new pathogen that causes blue mold on tomato plants in Japan.     

Vascular blackening of wasabi rhizomes caused by <Emphasis Type="Italic">Pectobacterium carotovorum</Emphasis> subsp. <Emphasis Type="Italic">carotovorum</Emphasis>

Wasabi (Wasabia japonica) is grown for its highly-valued rhizome which is used as a condiment in Japanese food. Symptoms of vascular blackening in the rhizome were first observed in 2005 in plants grown in British Columbia, Canada. Microscopic observations and microbial isolation from infected tissues revealed that most of the xylem tracheid cells were blackened and bacteria were consistently associated with symptomatic plants. The bacterium most frequently recovered was identified as Pectobacterium carotovorum subsp. carotovorum (Pcc) using BioLog™ and sequencing of a specific ~510 bp IGS region. Pathogen-free plants obtained using meristem-tip micropropagation were inoculated with a wasabi isolate of Pcc. Vascular blackening symptoms developed in the rhizome after 8 weeks when the rhizome was first wounded by stabbing or cutting, or if the roots were pre-inoculated with Pythium species isolated from rhizome epidermal tissues, followed by inoculation with Pcc at 1 × 10⁸ cells ml⁻¹. Xylem tracheid cells were blackened and Pcc was reisolated from all diseased tissues. The highest frequency of rhizome vascular blackening occurred at 22°C and 27°C and these tissues occasionally succumbed to soft rot at higher temperatures, but not when inoculated tissues were incubated at 10°C. The rooting medium used by growers for vegetative propagation of wasabi was shown to contain Pcc but the pathogen was not recovered from the irrigation water. Entry of Pcc through wounds on wasabi rhizomes and the host tissue response result in symptoms of vascular blackening.     

<Emphasis Type="Italic">Bchex</Emphasis> virulence gene of <Emphasis Type="Italic">Botrytis cinerea</Emphasis>: characterization and functional analysis

We previously identified Bchex as a highly expressed gene during filamentous growth in Botrytis cinerea. The gene encodes the principal protein of the Woronin body and has been shown to seal septal pores in response to cellular damage. In the present study, Southern blot analysis of genomic DNA indicated that the gene exists as a single copy in the B. cinerea genome. The gene was differentially expressed during various developmental stages: expression was high in germinating conidia and the mycelial stage and lower in resting conidia and the appressorial stage. For functional analyses, homologous recombination was used to obtain a ΔBchex knockout mutant. Growth of the mutant was strongly reduced growth in complete medium and in defined media with sucrose, fructose or pectin as the carbon source. After detached tomato leaves were inoculated with the Bchex mutant, lesion development was markedly reduced compared to the control, suggesting that Bchex participates in normal growth, germination and virulence of this fungus.     

Exogenous coronatine,but not coronafacic acid or methyl jasmonate,restores the disease phenotype of a coronatine-defective mutant of <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> pv. <Emphasis Type="Italic">tomato</Emphasis> on tomato seedlings

Coronatine (COR) functions as a virulence factor in the interaction of Pseudomonas syringae pv. tomato strain DC3000 with tomato and Arabidopsis. COR consists of two moieties, coronafacic acid (CFA) and coronamic acid (CMA). Both COR and CFA function as structural and functional analogues of jasmonic acid (JA) and related signaling compounds such as methyl jasmonate (MeJA) and JA-isoleucine (JA-Ile). The precise function of COR and whether MeJA functions as an analogue of COR in disease development are not known. In this study, we analyzed whether the COR-defective mutant DB29, which is a CFA⁻ CMA⁻ mutant of DC3000, could be complemented for virulence via the exogenous application of COR, CFA, or MeJA. When tomato seedlings were inoculated with DB29 and supplemented with exogenous COR, the DB29 population multiplied in tomato seedlings and induced disease phenotypes similar to wild-type DC3000. Although the addition of exogenous MeJA or CFA enhanced the multiplication of DB29, wild-type disease phenotypes could not be restored with these compounds. Furthermore, inoculation of DB29 along with exogenous COR, but not MeJA or CFA, suppressed the expression of defense-related genes and increased the accumulation of reactive oxygen species, which are associated with severe chlorosis. Taken together, our results suggest that although COR targets the jasmonate pathway by mimicking JA, the function of COR in disease development is distinctly different from MeJA or CFA.     

Transmission of <Emphasis Type="Italic">Tomato chlorotic dwarf viroid</Emphasis> by bumblebees (<Emphasis Type="Italic">Bombus ignitus</Emphasis>) in tomato plants

Quantitative PCR revealed that Tomato chlorotic dwarf viroid (TCDVd) was present in substantial amounts in viroid-infected tomato flowers. Healthy tomato plants were arranged in two different glasshouses, and plants were mechanically inoculated with TCDVd. Bumblebees (Bombus ignitus) were then introduced into the glasshouses to reveal whether the viroid was transmitted from infected source plants to neighbouring healthy plants. TCDVd infection was found in neighbouring tomato plants more than 1 month after the introduction of the bees, some of which expressed symptoms, in both glasshouses. Thus, bumblebees transmitted TCDVd from tomato to tomato by pollination activities.     

Cloning of the pathogenicity-related gene <Emphasis Type="Italic">FPD1</Emphasis> in <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">lycopersici</Emphasis>

We selected a reduced-pathogenicity mutant of Fusarium oxysporum f. sp. lycopersici, a tomato wilt pathogen, from the transformants generated by restriction enzyme-mediated integration (REMI) transformation. The gene tagged with the plasmid in the mutant was predicted to encode a protein of 321 amino acids and was designated FPD1. Homology search showed its partial similarity to a chloride conductance regulatory protein of Xenopus, suggesting that FPD1 is a transmembrane protein. Although the function of FPD1 has not been identified, it does participate in the pathogenicity of F. oxysporum f. sp. lycopersici because FPD1-deficient mutants reproduced the reduced pathogenicity on tomato.The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession number AB110097     

Dry mycelium of<Emphasis Type="Italic">Penicillium chrysogenum</Emphasis> protects cucumber and tomato plants against the root-knot nematode<Emphasis Type="Italic">Meloidogyne javanica</Emphasis>

Incorporation into soil of dry mycelium ofPenicillium chrysogenum, a waste product of the pharmacological industry, enhanced plant growth and reduced root galling caused by the root-knot nematodeMeloidogyne javanica in cucumber and tomato plants. Incorporation into sandy loam soil in pots of dry mycelium at a concentration of 0.25% (w/w) resulted in complete protection of cucumber plants from the nematode. The number of juveniles recovered from soils containing dry mycelium was greatly reduced even at a concentration of 0.1% (w/w). In microplot studies conducted at two sites in two seasons, with three or four doses, dry mycelium caused a dose-dependent reduction in root galling index (GI) and promotion of plant growth of cucumber and tomato plants. Inin vitro studies, the water extract of dry mycelium immobilized nematode juveniles and reduced the egg hatching rate, but these effects were partly reversible after a rinse in water. Soil-drenching of cucumber and tomato seedlings with water extract of dry mycelium did not reduce GI or number of root-invading juveniles. The results show that dry mycelium promotes plant growth and protects plants against nematode infection. Protection, however, does not operatevia induced resistance. http://www.phytoparasitica.org posting April 6, 2003.     

Generation and characterisation of Tn5-tagged <Emphasis Type="Italic">Xanthomonas oryzae</Emphasis> pv. <Emphasis Type="Italic">oryzae</Emphasis> mutants that overcome <Emphasis Type="Italic">Xa23</Emphasis>-mediated resistance to bacterial blight of rice

Xanthomonas oryzae pv. oryzae causes bacterial blight of rice. Xa23, a bacterial blight resistance gene identified originally in wild rice, Oryza rufipogon, is dominant and resistant to all X. oryzae pv. oryzae field isolates tested. The corresponding avirulence gene avrXa23 is unknown. Here we report the generation of a random insertion mutant library of X. oryzae pv. oryzae strain PXO99 using a Tn5-derived transposon tagging system, and identification of mutant strains that are virulent on CBB23, a near-isogenic rice line containing Xa23. A total of 24,192 Tn5 inserted clones was screened on CBB23 by leaf-cutting inoculation and at least eight of them caused lesions on CBB23 comparable to those on JG30, the susceptible recurrent parent of CBB23. Polymerase chain reaction and Southern blot analysis showed that all the eight mutants, designated as P99M1, P99M2, P99M3, P99M4, P99M5, P99M6, P99M7 and P99M8, have a single Tn5-insertion in their genomes. The flanking DNA sequences of the Tn5-insertion sites were isolated by PCR-walking and sequenced. Bioinformatic analysis of the flanking sequences, by aligning them with the whole genome sequences of X. oryzae pv. oryzae strains PXO99, KACC10331 and MAFF311018 through NCBI, revealed that the Tn5-insertions disrupted genes that encode TAL effector AvrBs3/PthA, ISXo1 transposase, Type II secretion system protein-like protein or outer membrane protein, glycogen synthase, cytochrome C5 and conserved hypothetical protein. Further identification of these mutants will facilitate the molecular cloning of avirulence gene avrXa23. The authors C.-L. Wang, A.-B. Xu contributed equally to this work; Y. Gao and Y.-L. Fan contributed equally to this work.     

Sensitivity of <Emphasis Type="Italic">Botrytis cinerea</Emphasis> to propamidine: in vitro determination of baseline sensitivity and the risk of resistance

The baseline sensitivity of Botrytis cinerea to propamidine and assessment of the risk of propamidine resistance in vitro are presented in this article. The baseline sensitivities of 41 wild-type strains were distributed as a unimodal curve with EC₅₀ values of mycelial growth ranging from 0.182 to 1.460 μg ml⁻¹, with a mean of 0.79 ± 0.27 μg ml⁻¹. A total of 10 resistant mutants, obtained from one parental strain, were induced by UV irradiation and selected for resistance to propamidine with an average frequency of 1.98 × 10⁻⁹ and 0.025 respectively. These mutants were divided into three classes of resistant phenotypes with low (LR), moderate (MR) and high (HR) levels of resistance, determined by the EC₅₀ values of 5.0–15.0 μg ml⁻¹, 15.1–75.0 μg ml⁻¹ and more than 75.0 μg ml⁻¹ respectively. Neither positive cross-resistance nor negative cross-resistance was detected between propamidine and the fungicides, benzimidazole carbendazim, anilino-pyrimidine pyrimethanil, dicarboximide iprodione or procymidone. All 10 propamidine-resistant mutants showed reduced mycelial growth in vitro, sporulation, spore germination and pathogenicity when compared with the parental strain. These studies demonstrated that propamidine possesses a low risk of resistance developing. However, as B. cinerea is a high-risk pathogen, appropriate precautions against resistance development should be taken.     

Effects of garlic (<Emphasis Type="Italic">Allium sativum</Emphasis>) juice containing allicin on <Emphasis Type="Italic">Phytophthora infestans</Emphasis> and downy mildew of cucumber caused by <Emphasis Type="Italic">Pseudoperonospora cubensis</Emphasis>

The volatile antimicrobial substance allicin (diallylthiosulphinate) is produced in garlic when the tissues are damaged and the substrate allicin (S-allyl-l-cysteine sulphoxide) mixes with the enzyme alliin-lyase (E.C.4.4.1.4). Allicin undergoes thiol-disulphide exchange reactions with free thiol groups in proteins and it is thought that this is the basis of its antimicrobial action. At 50 μg ml^-1, allicin in garlic juice inhibited the germination of sporangia and cysts and subsequent germ tube growth by Phytophthora infestans both in vitro and in vivo on the leaf surface. Disease severity in P. infestans-infected tomato seedlings was also reduced by spraying leaves with garlic juice containing allicin over the range tested (55–110 μg ml⁻¹) with an effectiveness ranging from approximately 45–100%. Similarly, in growth room experiments at concentrations from 50–1,000 μg ml⁻¹, allicin in garlic juice reduced the severity of cucumber downy mildew caused by Pseudoperonospora cubensis by approximately 50–100%. These results suggest a potential for developing preparations from garlic for use in specialised aspects of organic farming, e.g. for reducing pathogen inoculum potential and perhaps for use under glass in horticulture.