Analysis of the relationship between parameters of resistance to <Emphasis Type="Italic">Fusarium</Emphasis> head blight and <Emphasis Type="Italic">in vitro</Emphasis> tolerance to deoxynivalenol of the winter wheat cultivar WEK0609 <Superscript>®</Superscript>

Fusarium head blight (FHB) symptom development, relative spikelet weight (RSW), fungal DNA (FDNA) and deoxynivalenol (DON) content of grain was assessed in the FHB resistant winter wheat cv. WEK0609 and the FHB susceptible cv. Hobbit sib, and among doubled haploid progeny lines (DHLs) developed from a cross between these cultivars. In addition, the relationship between FHB resistance traits and germination on DON-containing medium (in vitro DON tolerance (IVDT)) was also investigated to assess the possibility of using this test as in vitro method of screening for FHB resistance in this cultivar. Analysis indicated that WEK0609 resistance significantly reduced symptom development, yield loss and the FDNA and DON content of grain relative to Hobbit sib. Although both the DON and FDNA content were greater in susceptible than in resistant progeny lines, the ratio of DON to FDNA decreased with increasing susceptibility. The resistance derived from WEK0609 appears to have a greater effect on colonisation of the grain by the fungus than on the accumulation of DON within the grain. In vitro tolerance to DON does not appear to relate to FHB resistance in WEK0609 and thus does not provide a means of selecting for FHB resistance derived from this cultivar.     

Effect of polyploidisation on the response of apple (<Emphasis Type="Italic">Malus</Emphasis> × <Emphasis Type="Italic">domestica</Emphasis> Borkh.) to <Emphasis Type="Italic">Venturia inaequalis</Emphasis> infection

Spinach is one of the most nutritious green-leaf vegetables. In the spinach production, diseases cause a significant loss in both yield and quality. Improving disease resistance is one of the major challenges in spinach breeding. Arabidopsis nucleoporin CONSTITUTIVE EXPRESSER OF PATHOGENESIS-RELATED GENES 5 (CPR5) functions as a negative regulator of plant cell death and immunity as cpr5 mutant exhibits spontaneous cell death and heightened immunity. In addition, CPR5 play a role in trichome development as the majority of cpr5 mutant trichomes are branchless whereas wild type trichomes are often three-branched. In the spinach genome, we identified a homolog of Arabidopsis CPR5, referred to as Spinacia oleracea CPR5 (SoCPR5). To investigate the function of SoCPR5, we introduced SoCPR5 into Arabidopsis cpr5 mutant. Our data showed that both spontaneous cell death and heightened immunity were suppressed in the SoCPR5-transgenic cpr5 mutants, verifying that SoCPR5 functions as its Arabidopsis counterpart in plant cell death and immunity. SoCPR5 also fully restored wild type trichome phenotype of the cpr5 mutant. Our study therefore indicates that the function of SoCPR5 is conserved between plant species and SoCPR5 can be applied for genetic manipulation of plant immunity in spinach.     

Does <Emphasis Type="Italic">Puccinia hemerocallidis</Emphasis> regularly host-alternate between <Emphasis Type="Italic">Hemerocallis</Emphasis> and <Emphasis Type="Italic">Patrinia</Emphasis> plants in Japan?

Daylily rust fungus, Puccinia hemerocallidis, was proven to host-alternate between a wild daylily, Hemerocallis fulva var. longituba, and a patrinia, Patrinia villosa. No proof was obtained for the early belief that the fungus is pathogenic to plantainlilies, Hosta species, in addition to daylilies, Hemerocallis species. The fungus seems to alternate regularly between daylilies and patrinias in Japan because most daylily species are deciduous, and a vegetatively reproducing stage of the pathogen does not seem capable of successfully overwintering free of the living host tissue.     

First report of ‘<Emphasis Type="Italic">Candidatus</Emphasis> Phytoplasma malaysianum’ associated with <Emphasis Type="Italic">Elaeocarpus</Emphasis> yellows of <Emphasis Type="Italic">Elaeocarpus zollingeri</Emphasis>

“Elaeocarpus yellows” (ELY) is a widely reported phytoplasma disease of Elaeocarpus zollingeri trees in Japan. The phytoplasma associated with ELY (ELY phytoplasma) had not been identified at the species level because its 16S rRNA sequence had yet to be reported. Here, we report the results of a sequence analysis based on 16S rRNA and secA gene sequences, which showed that the ELY phytoplasma is related to ‘Candidatus Phytoplasma malaysianum’. To our knowledge, this is the first report showing the occurrence of ‘Ca. P. malaysianum’ outside Malaysia and the infection of E. zollingeri by the phytoplasma.     

Activity of <Emphasis Type="Italic">ß</Emphasis>-1,3-glucanase and <Emphasis Type="Italic">ß</Emphasis>-1,4-glucanase in two potato cultivars following challenge by the fungal pathogen <Emphasis Type="Italic">Alternaria solani</Emphasis>

Early blight of potato, caused by Alternaria solani, is a ubiquitous disease in many countries around the world. Our previous screening of several Iranian potato cultivars found that variation in resistance exists between two cultivars: ‘Diamond’ and ‘Granula’. Cultivar Diamond is more resistant to multiple isolates of A. solani when compared to cv. Granula. Furthermore, we have found that different pathogen isolates have varying degrees of infection. We monitored the activities of two pathogen-related (PR) glucanase proteins in Diamond and Granula in response to two isolates of A. solani with different degrees of virulence. ß-1,3-glucanase and ß-1,4-glucanase activities were recorded in healthy and diseased leaves of potatoes up to 10 days after inoculation. Their activities were found to be higher in diseased leaves when compared to those of uninfected leaves. Our data suggest that significantly reduced activities of theses enzymes in potato could be related to a lower degree of resistance or an increased ability of a more aggressive isolate to suppress PR protein expression.     

<Emphasis Type="Italic">Amaranthus</Emphasis> spp.: a new host of “<Emphasis Type="Italic">Candidatus</Emphasis> Phytoplasma aurantifolia”

Mexico is considered to be one of the centers of origin of grain amaranth species. Recently, plants with abnormal anatomical features were observed in experimental fields established in Central Mexico. The most noticeable symptoms, which consisted of excessive stem and bud proliferation, mosaics and unusual coloration, suggested that they might be phytoplasma-induced disorders. Thus, different accessions of grain amaranth (Amaranthus hypochondriacus and A. cruentus) plants were subjected to polymerase chain reaction (PCR) analysis specifically designed to detect these pathogens. Two universal phytoplasma-specific primer pairs were tested in a nested PCR assay, with primer pair P1/tint (followed by primer pair R16F2/R16R2). Further DNA sequencing analysis of the resulting amplicons indicated that these phytoplasmas may be related to others already affecting important agricultural crops in Mexico, such as soybean. Data are presented that disclose the etiology of these syndromes by the use of molecular techniques. To the best of our knowledge, this finding constitutes the first report of a phytoplasma-related disease in grain amaranth.     

PCR-mediated Detection of <Emphasis Type="Italic">Xanthomonas oryzae </Emphasis>pv. <Emphasis Type="Italic">oryzae </Emphasis>by Amplification of the 16S–23S rDNA Spacer Region Sequence

A detection method specific for Xanthomonas oryzae pv. oryzae, the pathogen responsible for bacterial blight of rice, was based on the polymerase chain reaction (PCR) and designed by amplifying the 16S–23S rDNA spacer region from this bacterium. The nucleotide sequence of the spacer region between the 16S and 23S rDNA, consisting of approximately 580-bp, from X. oryzae pv. oryzae, X. campestris pv. alfalfae, X. campestris pv. campestris, X. campestris pv. cannabis, X. campestris pv. citri, X. campestris pv. cucurbitae, X. campestris pv. pisi, X. campestris pv. pruni and X. campestris pv. vitians, was determined. The determined sequences had more than 95% identity. Therefore, a pair of primers, XOR-F (5′-GCATGACGTCATCGTCCTGT-3′) and XOR-R2 (5′-CTCGGAGCTATATGCCGTGC-3′) was designed and found to specifically amplify a 470-bp fragment from all strains of X. oryzae pv. oryzae isolated from diverse regions in Japan. No PCR product was amplified from X. campestris pathovars alfalfae, campestris, cannabis, carotae, cucurbitae, dieffenbachiae, glycines, pisi, pruni, vitians or zantedeschiae, except for pathovars citri, incanae and zinniae. The method could also detect the pathogen in infected rice leaves within 3 hr, at a detection limit of 4×10¹ cfu/ml. Received 17 December 1999/ Accepted in revised form 10 April 2000     

Transgenic citrus expressing the antimicrobial gene <Emphasis Type="Italic">Attacin E</Emphasis> (<Emphasis Type="Italic">attE</Emphasis>) reduces the susceptibility of ‘Duncan’ grapefruit to the citrus scab caused by <Emphasis Type="Italic">Elsinoë fawcettii</Emphasis>

Citrus scab, caused by Elsinoë fawcettii (anamorph Sphaceloma fawcettii), is a common foliar fungal disease affecting many citrus cultivars, including grapefruit. No commercial grapefruit cultivar is resistant to scab, and the disease results in severely blemished fruit which reduces its marketability. Transgenic ‘Duncan’ grapefruit trees expressing the antimicrobial attE gene were produced via Agrobacterium-mediated transformation. In in vitro leaf and greenhouse assays, several transgenic-lines had significantly lower susceptibility to E. fawcettii compared to the non-transformed control (P?P?P?attE mRNA was inversely related to the number of copies detected by Southern blot. The least susceptible line had a single inserted copy of the attE transgene whereas more susceptible lines had multiple copies. Since the attacin mode of action was thought to be specific to Gram-negative bacteria, it was unexpected to find that there was a significant activity against E. fawcettii.     

<Emphasis Type="Italic">Spiraea salicifolia:</Emphasis> a new plant host of “<Emphasis Type="Italic">Candidatus</Emphasis> Phytoplasma ziziphi”-related phytoplasma

Spiraea salicifolia is widely grown in China as an ornamental plant, and its roots and young leaves have many medical uses. On the campus of Northwest A&F University, we observed diseased S. salicifolia plants that had yellowed, dwarfed, deformed leaves and other symptoms resembling diseases caused by phytoplasma. This study was aimed at determining the causal agent of the disease. On the basis of phytoplasma-specific DNA amplification by PCR, a phytoplasma infection of S. salicifolia was confirmed. The phytoplasma was related to “Ca. Phytoplasma ziziphi” according to RFLP and phylogenetic analyses. This report is the first of phytoplasma infection of S. salicifolia in China.     

The first detection of ‘<Emphasis Type="Italic">Candidatus</Emphasis> Phytoplasma trifolii’ in <Emphasis Type="Italic">Rhododendron</Emphasis> hybridum

Four Rhododendron hybridum plants (from cvs Moravanka and Don Juan), all exhibited symptoms of shortened axillary shoots, reduced leaves with vein clearing and yellowing, undeveloped flowers, and general stunting in a rhododendron nursery garden in southern Bohemia in 2007. Electron microscopy examination of ultra-thin sections revealed the presence of numerous polymorphic phytoplasma-like bodies in the phloem tissue of leaf midribs and petioles. The phytoplasma etiology of this disease was further confirmed by polymerase chain reaction (PCR) using universal phytoplasma primers. Restriction fragment length polymorphism (RFLP) analysis of amplification products obtained with a R16F2/R16R2 primer pair from all symptomatic plants indicated the presence of phytoplasma from the 16SrVI-A subgroup. A detailed comparison of the amplified sequences and phylogenetic analysis confirmed that the phytoplasma belonged to the subgroup 16SrVI-A (clover proliferation phytoplasma group). This is the first report of the natural occurrence of ‘Candidatus Phytoplasma trifolii’ in plants of Rhododendron hybridum.     

Pathogenicity and Fungicide Sensitivity of <Emphasis Type="Italic">Rhizoctonia solani</Emphasis> and <Emphasis Type="Italic">R. cerealis</Emphasis> Isolates

The Rhizoctonia solani species consists of multinucleate isolates that belong to anastomosis groups AG1–AG3 and differ in virulence and host affinity. R. cerealis is a binucleate species of anastomosis group AG-D which causes sharp eyespot, a common plant disease in Poland. Rhizoctonia spp. is a ubiquitous soil pathogen that poses a significant threat for global crop production due to the absence of effective crop protection products. The aim of this study was to determine the virulence of R. solani and R. cerealis isolates towards Beta vulgaris, Zea mays, Triticum spelta and T. aestivum seedlings, to confirm the presence of endopolygalacturonase genes pg1 and pg5 in the genomes of the tested isolates and to evaluate the tested isolates’ sensitivity to triazole, strobilurin, imidazole and carboxamide fungicides. All tested isolates infected B. vulgaris seedlings. but none of them were virulent against Z. mays plants. R. solani isolates AG4 PL and AG2-2IIIB PL were characterized by the highest virulence (average infestation score of 2.37 and 2.53 points on a scale of 0–3 points) against sugar beet seedlings. The prevalence of infections caused by most of the analysed isolates (in particular R. solani AG4 J—11.8, and R. cerealis RC2—0.78) was higher in spelt than in bread wheat. The virulence of the analysed isolates was not correlated with the presence of pg1 and pg5 genes. The efficacy of the tested fungicides in controlling Rhizoctonia spp. infections was estimated at 100% (propiconazole + cyproconazole), 98.8% (penthiopyrad), 95.4% (tebuconazole) and 78.3% (azoxystrobin).     

<Emphasis Type="Italic">Talaromyces wortmannii</Emphasis> FS2 emits <Emphasis Type="Italic">β</Emphasis>-caryphyllene,which promotes plant growth and induces resistance

A plant-growth-promoting fungus (PGPF), Talaromyces sp. was isolated from an agricultural field in southwestern Japan. We found that this fungus emitted several terpenoid-like volatiles including β-caryophyllene. Then we investigated the effect of β-caryophyllene on promoting the growth and inducing resistance of Brassica campestris L. var. perviridis. The compound significantly enhanced the growth of seedlings and their resistance to Colletotrichum higginsianum. On the basis of these results, we discuss the role of β-caryophyllene in the activities of PGPF.     

Mechanisms of induced resistance in lettuce against <Emphasis Type="Italic">Bremia lactucae</Emphasis> by DL<Emphasis Type="Bold">-</Emphasis>β<Emphasis Type="Bold">-</Emphasis>amino<Emphasis Type="Bold">-</Emphasis>butyric acid (BABA)

BABA induced local and systemic resistance in lettuce (Lactuca sativa) against the Oomycete Bremia lactucae. Structure-activity analysis showed no induced resistance by related amino-butanoic acids or β-alanine. The R-enantiomer of BABA induced resistance whereas the S-enantiomer did not, suggesting binding to a specific receptor. Other compounds known to be involved in SAR signaling, including abscisic acid, methyl-jasmonate, ethylene, sodium-salicylate and Bion® (BTH) did not induce resistance. Systemic translocation of ¹⁴C-BABA and systemic protection against downy mildew were tightly correlated. BABA did not affect spore germination, appressorium formation, or penetration of B. lactucae into the host. Epifluorescence and confocal microscopy revealed that BABA induced rapid encasement with callose of the primary infection structures of the pathogen, thus preventing it from further developing intercellular hyphae and haustoria. Invaded host cells treated with BABA did not accumulate phenolics, callose or lignin, or express HR. In contrast, cells of genetically-resistant cultivars accumulated phenolics, callose and lignin and exhibited HR within one day after inoculation. The callose synthesis inhibitor DDG did not inhibit callose encasement nor compromised the resistance induced by BABA. PR-proteins accumulated too late to be responsible for the induced resistance. DAB staining indicated that BABA induced a rapid accumulation of H₂O₂ in the penetrated epidermal host cells. Whether H₂O₂ stops the pathogen directly or via another metabolic route is not known.     

Detection of <Emphasis Type="Italic">Phoma valerianellae</Emphasis> in lamb’s lettuce seeds

After the reappearance in Italy of a foliar disease of lamb’s lettuce (Valerianella olitoria L. Poll. syn. V. locusta L. Betcke) incited by Phoma valerianellae, we set out to measure the level of seed infection by this fungus, using a plating test, and to develop a molecular method for quick and reliable detection of the pathogen in seeds. All six samples of lamb’s lettuce seed tested were contaminated by P. valerianellae at levels of 0.6% to 15%. Surface disinfection of seeds did not eliminate the contamination and only reduced it to between 0.1% and 10%. The need for a sensitive, reliable and rapid diagnostic method for early identification of the fungus exists. We have developed a PCR-based method to identify the fungus in seeds. Variation within the internal transcribed spacer (ITS1, 5.8S sequences and ITS2) region of the rDNA (ITS) was used to characterize the P. valerianellae strains and to design specific primers within the ITS region.     

Identification of a novel point mutation in the <Emphasis Type="Italic">β</Emphasis>-tubulin gene of <Emphasis Type="Italic">Botrytis cinerea</Emphasis> and detection of benzimidazole resistance by a diagnostic PCR-RFLP assay

The molecular basis of resistance to benzimidazole fungicides with laboratory and field mutant isolates of Botrytis cinerea was investigated. After chemical mutagenesis with N-methyl-N-nitrosogouanidine (NMNG) two different benzimidazole-resistant phenotypes were isolated on media containing carbendazim or a mixture of carbendazim and diethofencarb. The mutant isolates from the fungicide-mixture-containing medium were moderately resistant to carbendazim with wild-type tolerance to diethofencarb while mutant isolates from carbendazim-containing medium were highly resistant to carbendazim but sensitive to diethofencarb. The studied field isolates were highly resistant to benzimidazoles and sensitive to diethofencarb. Study of fitness characteristics of benzimidazole highly-resistant isolates showed that the resistance mutation(s) had no apparent effect on fitness-determining parameters. Contrary to this, the moderately benzimidazole-resistant strains, with no increased diethofencarb sensitivity, had a significant reduction in certain ecological fitness-determining characteristics. Analysis of the sequence of the β-tubulin gene revealed two amino acid replacements in the highly benzimidazole-resistant mutants compared to that of the wild-type parent strain. One was the glutamic acid (GAG) to alanine (GCG) change at position 198 (E198A), identified in both laboratory and field highly benzimidazole-resistant isolates, a mutation previously implicated in benzimidazole resistance. The second was a novel benzimidazole resistance mutation of glutamic acid (GAG) to glycine (GGG) substitution at the same position 198 (E198G), identified in a highly benzimidazole-resistant laboratory mutant strain. Molecular analysis of the moderately benzimidazole-resistant strains revealed no mutations at the β-tubulin gene. A novel diagnostic PCR-RFLP assay utilising a BsaI restriction site present in the benzimidazole-sensitive (E198) but absent in both resistant genotypes (E198G and E198A) was developed for the detection of both amino acid replacements at the β-tubulin gene.     

Is there a relationship between the intrinsic rate of propagation and <Emphasis Type="Italic">in-vitro</Emphasis> migration and virulence of the pinewood nematode, <Emphasis Type="Italic">Bursaphelenchus xylophilus</Emphasis>?

We investigated if rates of propagation and migration were related with the level of virulence in the pinewood nematode Bursaphelenchus xylophilus using 17 offspring lines from the F₂ crosses between virulent and avirulent isolates. Virulence was tested by inoculating seedlings of Pinus thunbergii with the nematodes. The proportion of dead seedlings ranged from 13.3% to 77.8%, 20 weeks after inoculation. Migration rate of the nematodes was estimated by measuring their migration distance per unit time in an artificial substrate that imitated pathways in pine trees. Migration rate varied from 0.85 to 3.53 mm min⁻¹. Propagation rate was determined based on population growth on the fungus Botrytis cinerea, and it ranged between 10^3.88 and 10^4.99 per 12 days. Statistical analyses revealed that virulence was not correlated with migration rate, but was negatively correlated with propagation rate on Botrytis cinerea, suggesting that the nematodes paid some cost for virulence. Also, there was no relationship between rates of migration and propagation. Cluster analysis showed that the biological parameters varied between crossbred lines, with no kinship bias, suggesting the absence of sex-linked inheritance in virulence and rates of propagation and migration.     

Detection of <Emphasis Type="Italic">Ageratum enation virus</Emphasis> from cat’s whiskers (<Emphasis Type="Italic">Cleome gynandra</Emphasis> L.) with leaf curl symptoms in India

An association of a begomovirus with leaf curl symptoms on Cleome gynandra was detected using a polymerase chain reaction (PCR) with begomovirus-specific primers. Further, the complete DNA-A of the begomovirus was cloned and sequenced. BLAST analysis of the sequence data revealed 92–99% identities and close relationships with several isolates of Ageratum enation virus (AgEV); therefore, we identified the virus associated with leaf curl symptoms of C. gynandra as an isolate of AgEV. This report is the first on the detection of AgEV in plants of C. gynandra with leaf curl in India.