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.     

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.     

Bois noir affects the yield and wine quality of <Emphasis Type="Italic">Vitis vinifera</Emphasis> L. cv. ‘Chardonnay’

The Bois noir (BN) disease induced by ‘Candidatus Phytoplasma solani’ (CPs) is common in European vineyards. Its damage has not been fully investigated, especially with regards to wine attributes. The impact of BN on yield, berry composition and wine characteristics of Vitis vinifera L. cv. ‘Chardonnay’ was therefore comprehensively characterized in a 3-year field experiment in Hungary, Eger winegrowing region. Additionally, the bindweed-related tuf-b1 genotype was identified to be involved in the BN pathosystem in the experimental vineyard. Infection of CPs tuf-b1 genotype resulted in severe yield loss, the average decrease in number of bunches and total yield per vine was 56.7% and 68.4%, respectively. Analyses of wines produced from grapes of BN infected vines revealed decreased alcohol, epicatechin and iron contents; and increased organic acids, titratable acidity, catechin and calcium contents. Sensory evaluation of these wines confirmed unfavourable characteristics, i.e. higher acidity, bitterness, and usually pinkish discolouration. Negative impact on berry composition and wine quality were pronounced in the vintage with favourable weather conditions for grapevine production, whereas the negative effects of BN infection were less prominent, even masked, in the vintages with unfavourable weather (wet and cool). To reduce BN-caused damage, the need for improved preventative and curative measures for BN disease is highlighted.     

<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.     

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     

A model system for plant-virus interaction—infectivity and seed transmission of <Emphasis Type="Italic">Cherry leaf roll virus</Emphasis> (CLRV) in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

The wide natural incidence of Cherry leaf roll virus (CLRV) in deciduous forest trees and nurseries in northern Europe is believed to have occurred, apart from occasional mechanical spread and transmission through grafting, mainly by seed transmission. The mode of the vertical transmission and its role in the epidemiology of the virus has not been investigated, basically due to the inconvenient host-pathogen combinations studied to date. With the aim of obtaining an appropriate system for identification of viral genes and products participating in infection processes and seed transmission of CLRV, we performed infection and seed transmissibility tests with CLRV in Arabidopsis thaliana plants. Two phylogenetically and serologically different CLRV isolates were tested. Both of them were found able to infect A. thaliana plants, exhibited clear symptoms of the infection and spread systemically in the plants. Infection of the seeds and of a remarkable number of seedlings generated from infected seeds was possible for two consecutive generations. These results, for first time, report seed transmission of CLRV in the model plant A. thaliana and allow the assumption to be made of embryo invasion during seed transmission. Furthermore, first indications are given that genetically diverse CLRV isolates exhibit different abilities for vertical transmission in A. thaliana. The CLRV-A. thaliana model system is suitable for investigating viral invasion of developing plant organs and meristematic tissue, a prerequisite for successful virus dissemination via vertical transmission through seed.     

Detection and identification of a ‘<Emphasis Type="Italic">Candidatus</Emphasis> phytoplasma aurantifolia’-related strain (16SrII-A subgroup) associated with <Emphasis Type="Italic">corchorus aestuans</Emphasis> phyllody in China

In September 2015, a phyllody that is typical of phytoplasma infection was observed on Corchorus aestuans plants in Haikou, Hainan Province, China. Total DNA from symptomatic and asymptomatic plants was extracted for molecular diagnosis. On the basis of sequence analysis and phylogenetic trees based on 16S rDNA and rp genes, the phyllody phytoplasma was ascertained to be related to ‘Candidatus phytoplasma aurantifolia’. To the best of our knowledge, this is the first report of a phytoplasma infecting C. aestuans in the world.     

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.     

Protective Role of a methanolic Extract of Spinach (<Emphasis Type="Italic">Spinacia oleracea</Emphasis> L.) Against Adverse Effects of UV-C Irradiation on Fenugreek (<Emphasis Type="Italic">Trigonella foenum-graecum</Emphasis> L.) Seedlings

Photosynthesis is an important process for plants in which plants utilize the energy from sunlight to manufacture and produce all their own food. Although UV-C damage is not physiologically relevant for plants growing in the sun, short-wavelength (UV-C) radiation from germicidal lamps has been used to study cell damage in animals as well as in plants. This study provides a review of the effects of a methanolic extract of spinach (SE) as a natural powerful antioxidants used at two concentrations (25 and 50?ppm) on fenugreek seedlings grown under UV-C stress conditions. The results revealed that exposure of fenugreek seedlings to low UV-C period (30?min) increased growth criteria of the produced seedlings. In addition, the contents of chlorophyll, total pigments, total protein, free amino acids, non-enzymatic (carotenoids, total phenol, flavonoids, α?tocopherol & ascorbic acid contents) and enzymatic antioxidants were increased at this period. By increasing exposure periods of UV-C radiation, the growth criteria and chemical constitutes of the seedlings were decreased. Application of lower concentration of SE (25?ppm) improve the growth of the seedlings at 30-min UV-C and was able to protect seedlings from high periods of UV-C irradiation by increasing all measured parameters except CAT. These increases might be an adaptive mechanism to minimize the adverse effects of longer exposure of UV-C radiation. Moreover, treating the irradiated seedlings with spinach extract led to an obvious alteration in gene expression including synthesis of some proteins bands and disappearance of other protein sets of the protein profile.     

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.     

Virulence structure of the <Emphasis Type="Italic">Blumeria graminis</Emphasis> DC.f. sp. <Emphasis Type="Italic">avenae</Emphasis> populations occurring in Poland across 2010–2013

The aim of the present study was to determine the virulence structure of powdery mildew of oats (Blumeria graminis DC.f. sp. avena) in Poland in the years 2010–2013. For this purpose, powdery mildew isolates were collected from three experimental stations in Poland. To assess the virulence of the isolates, eight oat varieties with different responses to the pathogen were used. The results showed that a significant proportion of powdery mildew isolates found in Poland overcame the resistance genes of varieties Bruno (Pm6), Jumbo (Pm1) and Mostyn (Pm3). In contrast, lines Av1860 (Pm4), Am27 (Pm5) and Cc3678 (Pm2) were completely resistant to all pathogen isolates involved in the experiment. Changes constantly occurring in the powdery mildew population perfectly reflect diversity indexes, which were the smallest in the first year of observation, where in the following years these parameters were significantly higher. It is worth noting that the presence of powdery mildew is seasonal and local, which is reflected in the prevalence of the disease in a defined area of the country.     

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.     

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.     

Induction of oxidants in tomato leaves treated with <Emphasis Type="SmallCaps">DL</Emphasis>-β-Amino butyric acid (BABA) and infected with <Emphasis Type="Italic">Clavibacter michiganensis</Emphasis> ssp. <Emphasis Type="Italic">michiganensis</Emphasis>

Bacterial canker is an economically important disease of tomato. Resistance induced by DL-β-Amino butyric acid against bacterial canker caused by Clavibacter michiganensis ssp. michiganensis in tomato plants was investigated. Different doses of DL-β-Amino butyric acid (250–1000 μg ml⁻¹ doses) were tested on 3-week old plants inoculated with a 10⁸ CFU ml⁻¹ bacterial suspension, and disease development was evaluated after inoculation and treatment. Although in vitro growth of the bacteria was not affected by DL-β-Amino butyric acid treatment, foliage sprays of 500 μg ml⁻¹ DL-β-Amino butyric acid significantly suppressed disease development up to 54% by day 14 after inoculation at the four different doses tested. Bacterial populations were reduced by 84% in BABA-treated plants compared to water-treated plants by day 4 after inoculation. Inoculated BABA-treated plants showed significantly higher phenylalanine ammonia-lyase activity, peroxidase activity, and H₂O₂ concentration than inoculated water-treated plants during day 1 after treatment. These findings suggest that the DL-β-Amino butyric acid treatment resulted in an increase of these enzymes and in H₂O₂ concentration in planta, and was associated with induction of resistance to bacterial canker.     

β-aminobutyric acid protects <Emphasis Type="Italic">Brassica napus</Emphasis> plants from infection by <Emphasis Type="Italic">Leptosphaeria maculans.</Emphasis> Resistance induction or a direct antifungal effect?

Resistance to infection in plants can be induced by treatment with various chemicals. One such compound is β-aminobutyric acid (BABA). Its positive effect on disease resistance has been noted in several pathosystems. Here we demonstrate that treatment with BABA protects Brassica napus plants from infection by the fungal pathogen Leptosphaeria maculans. Surprisingly, BABA also displayes in vitro antifungal activity against L. maculans with EC₅₀ similar to the fungicide tebuconazole. Both spore germination and hyphal growth were affected. The toxic effect can be reverted by addition of trypton to the culture medium. We hypothesised that BABA might inhibit inorganic nitrogen assimilation. Suppression of disease progression in plants and antifungal activity in vitro was weaker for α-aminobutyric acid and negligible for γ-aminobutyric acid. In contrast to a resistance inducer benzothiadiazole, the effect of BABA on disease development was nearly independent of the timing of treatment, indicating possible antifungal activity in planta. On the other hand, quantification of multiple hormones and an expression analysis have shown that treatment with BABA induces a synthesis of salicylic acid (SA) and expression of SA marker gene PR-1, but no evidence was observed for priming of SA responses to L. maculans. While we have not conclusively demonstrated how BABA suppresses the disease progression, our results do indicate that antifungal activity is another mechanism by which BABA can protect plants from infection.     

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).     

Identification of some Fruit Characteristics in Wild Bilberry (<Emphasis Type="Italic">Vaccinium myrtillus</Emphasis> L.) Accessions from Eastern Anatolia

Some important physicochemical and bioactive characteristics of disease free 10 wild grown bilberry (Vaccinium myrtillus) accessions were evaluated. External and internal fruit quality was assessed by standard parameters (fruit weight, fruit color, fruit firmness, soluble solids, pH and total acidity) and bioactive contents (total phenolics, total anthocyanins, total antioxidant capacity and, vitamin C) in fruit were also determined. The commercial grown northern higbush blueberry, Vaccinium corymbosum cv. Bluecrop also included in the study to make comparision with bilberry samples. The highbush blueberry cv. Bluecrop had distinctive external fruit characteristics, such as bigger and more attractive fruits. However, the wild grown bilberry accessions showed interesting characters in mesocarp, such as high total phenolic content, total anthocyanin and total antioxidant capacity. Total phenolic and total anthocyanin content was 327?mg gallic acid equivalent and 142?mg of cyanidin-3-glucoside equivalent in 100?g fresh fruit in cv. Bluecrop while it was between 576–624?mg gallic acid equaivalent and 296–324?mg of cyanidin-3-glucoside equivalent in 100?g fresh fruits of bilberry accessions. Moreover, wild accessions approximately had 2 folds higher antioxidant capacity than cv. Bluecrop. Results suggested that bilberry accessions may serve as a source of desirable genes to develop improved varieties that respond to the new needs of the market.