Differentially-regulated defence genes in Malus domestica during phytoplasma infection and recovery

To improve knowledge about plant/phytoplasma interactions and, in particular, about the ‘recovery’ phenomenon in previously-infected plants, we investigated and compared expression levels of several defence-related genes (four pathogenesis-related proteins and three jasmonate-pathway marker enzymes) in apple plants showing different states of health: vigorous (healthy), phytoplasma-infected, and recovered. Real Time-PCR analyses demonstrated that genes are differentially expressed in apple leaf tissue according to the plants’ state of health. Malus domestica Pathogenesis-Related protein (MdPR) 1, MdPR 2 and MdPR 5 were significantly induced in leaves of diseased and symptomatic plants compared to leaves of those plants that were healthy or recovered. On the other hand, levels of all the jasmonate (JA)-pathway marker genes that we selected for this study, were up-regulated in the leaves of recovered plants compared to the diseased ones. In conclusion, our study demonstrated that two different sets of defence genes are involved in the interactions between apple plants and ‘Candidatus Phytoplasma mali’ (‘Ca. P. mali’) and that these genes are differentially expressed during phytoplasma infection or recovery.     

Effects of stolbur phytoplasma infection on DNA methylation processes in tomato plants

DNA methylation was investigated as a possible mechanism for regulation of floral gene expression in stolbur phytoplasma‐infected tomato buds. Expression of methylase and demethylase genes was found to be globally down‐regulated in tomato plants infected with stolbur isolate PO, but not in those infected with isolate C. These results are consistent with the finding that SlDEF, a gene orthologous to arabidopsis APETALA3 which is involved in petal formation, was down‐regulated in stolbur PO‐infected buds and remained unaffected in stolbur C‐infected buds, and with the fact that the two stolbur phytoplasma isolates C and PO induce distinct symptoms. Because of variations between the different cell‐types of the flower buds, the DNA methylation status of SlDEF could not be clearly established. However, the finding that treatment of stolbur PO‐infected plants with 5‐azacytidine partially restored SlDEF gene expression strongly suggests that DNA methylation is involved in down‐regulation of floral development genes in stolbur PO‐infected tomatoes.     

Expression of engineered antibodies in plants: a possible tool for spiroplasma and phytoplasma disease control

ABSTRACT In an attempt to develop maize plants with resistance to corn stunt spi-roplasma (CSS), a single-chain Fv fragment (scFv) gene that was constructed from antibodies with strong inhibitory activity against CSS, was expressed in a cell line of maize. However, plants regenerated from this transgenic cell line showed no distinct resistance to CSS infection under the greenhouse conditions. The affinity and functionality of scFv in vivo and the locations of CSS and expressed scFv in maize could be a part of the contributing factors affecting this result. Progress of expressing antibodies in plants for plant pathogen resistance is also discussed.     

Detection of lethal yellowing phytoplasma in embryos from coconut palms infected with Cape St Paul wilt disease in Ghana

This study investigated the potential of seed transmission of Cape St. Paul wilt disease (CSPWD) in coconuts. PCR amplification was used to assess the distribution of phytoplasmas in parts of West African Tall (WAT) palms infected with CSPWD. Employing phytoplasma universal primer pair P1/P7 in standard PCR, or followed with a nested PCR using CSPWD–specific primer pair G813f/AwkaSR, phytoplasma infection was detected in the trunks, peduncles, spikelets, male and female flowers of four infected WAT coconut palms. Through nested PCR, phytoplasma was also detected in four of 19 embryo DNA samples extracted individually from fruits harvested from three of the four infected palms and was confirmed as CSPWD by cloning and sequencing. Subsequently, CSPWD phytoplasma was again detected in five of 33 embryos from nine infected palms, and in one of eight fruits from two symptomless palms. Fruits from infected palms recorded higher percentage germinations in two field nurseries (average of 71·0%) compared to fruits from healthy palms (average of 57·6%), and matured fruits that had dropped from infected palms showed the same levels of germination as those harvested directly from the palms. This indicates that infected fruits retain the ability to germinate whether harvested or dropped. No phytoplasmas were detected in any of the resulting seedlings and plantlets obtained through embryo in-vitro culture. Therefore, although phytoplasma DNA can be detected in embryos, there is as yet no evidence that the pathogen is seed transmitted through to the seedling to cause disease in progeny palms.     

Expression patterns of defence genes and antioxidant defence responses in a rice variety that is resistant to leaf blast but susceptible to neck blast

The rice stage resistance variety Jiajing3768 is resistant to leaf blast but susceptible to neck blast. This variety was used to analyze the expression patterns of defence genes and antioxidant defence responses in the leaves at the seedling stage (LSS) and necks at the preliminary heading stage (NPHS), after inoculation with Magnaporthe oryzae. We found that defence genes PR1b (pathogenesis-related class 1b), PR4, PR10a, JIOsPR10 (jasmonic acid induced rice PR 10), Gns1 (1,3; 1,4-β-glucanase), Cht-1 (chitinase), and LOX (lipoxygenase) may play roles in the stage resistance in Jiajing3768; PAL (phenylalanine ammonia-lyase) and CHS (chalcone synthase) could participate in defending Jiajing3768 against neck blast but not leaf blast. The antioxidant enzymes superoxide dismutase, peroxidase, catalase, glutathione reductase, and malondialdehyde coordinately participate in the stage resistance to blast in Jiajing3768, and that oxidative damage is less in the LSS than in the NPHS.     

Recovery in apple trees infected with the apple proliferation phytoplasma: an ultrastructural and biochemical study

ABSTRACT Localization of hydrogen peroxide (H(2)O(2)) and the roles of peroxidases, malondialdehyde, and reduced glutathione in three apple cultivars were compared in healthy trees, trees infected with apple proliferation phytoplasma (APP), and trees that had recovered from the infection. In recovered apple trees, symptoms of the disease and the pathogen had disappeared from the canopy, but phytoplasmas remained in the roots. H(2)O(2) was detected cytochemically by its reaction with cerium chloride to produce electron-dense deposits of cerium perhydroxides.H(2)O(2) occurred in the plasmalemma of the phloem of leaves of recovered apple trees, but not in healthy or APP-infected leaves. In all cultivars, the peroxidase activity detected in tissue from APP-diseased trees was greater than or equal to that of tissue from recovered trees, which equaled or exceeded that of tissue from healthy trees, at two sampling times (May and September). In contrast, the glutathione content of leaves decreased in the reverse order. More malondialdehyde was observed in leaves from recovered trees than in leaves from healthy or APP-infected trees in three of six cultivar-date combinations; in the other three combinations, the malondialdehyde contents of leaves from healthy, infected, and recovered trees were not significantly different from one another. The results suggest that some components of the oxidant-scavenging system in recovered leaves are not very active, leading to an overproduction of H(2)O(2) and, possibly, to a membrane lipid peroxidation.The production of H(2)O(2) appears to be involved in counteracting pathogen virulence.     

Flavescence dorée phytoplasma titre in field‐infected Barbera and Nebbiolo grapevines

Flavescence dorée phytoplasma (FDP) titre in two red grapevine cultivars, Barbera and Nebbiolo, was measured over the vegetative seasons of two consecutive years in two vineyards of the Piemonte Region (northwestern Italy), with a double absolute quantification of FDP cells and grapevine DNA in real‐time PCR. The relationships of pathogen concentration to cultivar susceptibility and symptom severity were investigated. FDP titre was always higher in cv. Barbera than in cv. Nebbiolo infected vines, and this difference was significant at early and late summer samplings of 2008 and at early summer sampling of 2009. A seasonal trend in FDP concentration (low in spring, high in early summer and intermediate in late summer) was conserved for cvs Barbera and Nebbiolo in both years and vineyards. Considering both cultivars and years from both vineyards, a significant positive correlation between FDP concentration and symptom severity was found in the spring samples. Regarding the FDP strains (‐C or ‐D), no differences in pathogen titres were detected for either cultivar. Similarly, the presence of another grapevine yellows phytoplasma, bois noir, a subgroup 16SrXII‐A phytoplasma, in mixed infection with FDP strains had no effect on FDP concentration. These results demonstrate for the first time that grapevine cultivars with different susceptibility to FDP support different pathogen titres.     

Roles of stolbur phytoplasma and <Emphasis Type="Italic">Reptalus panzeri</Emphasis> (Cixiinae,Auchenorrhyncha) in the epidemiology of Maize redness in Serbia

Maize redness (MR), a disease causing midrib, leaf and stalk reddening and abnormal ear development in maize, has been reported from Serbia, Romania and Bulgaria for 50 years. Recent epiphytotics reduced yields by 40%–90% in southern Banat, Serbia. MR was recently associated with the presence of the stolbur phytoplasma, although the epidemiology of the disease remained unknown. Diseased fields in southern Banat were surveyed for potential vectors of the phytoplasma during 2005 and 2006, and high populations of Reptalus panzeri were found. In affected fields, 20% of the R. panzeri individuals and 85% of symptomatic maize plants carried the stolbur phytoplasma. When stolbur phytoplasma-infected R. panzeri were introduced into insect-free mesh cages containing healthy maize plants, midrib and leaf reddening developed on 48% of plants and stolbur phytoplasma was detected in 90% of the symptomatic plants. No symptoms or phytoplasma-positive plants were found in cages without insects. These data indicate that MR symptoms are associated with the stolbur phytoplasma. Reptalus panzeri is both abundant in affected fields and can transmit the stolbur phytoplasma, indicating the insect is likely to be a major vector of MR.     

Effect of soil temperature on disease development in melon plants infected by Monosporascus cannonballus

The effect of soil temperature on melon collapse induced by Monosporascus cannonballus was studied in the laboratory and in the field. In the laboratory, ascospore germination and hyphal penetration into melon roots were enhanced by increasing the temperature from 20 to 32°C. The optimum temperature for mycelial growth of five isolates of M. cannonballus was 30°C. In the field, the effect of temperature was studied in experiments conducted during the winter and autumn cropping seasons from 1995 to 1998. Disease progress was much faster in the autumn than in the winter crop seasons. Disease incidence reached 100% in the three consecutive autumn seasons studied. In the winter seasons, however, planting date influenced disease incidence. Early planting, at the beginning of January, resulted in a low disease incidence (6–26%, 125 days after planting), whereas planting at the end of January resulted in higher disease incidence (72–88%, 95–119 days after planting). In plots in which the soil was artificially heated to 35°C during the winter season, disease incidence reached 85%, as in the autumn season. Plants grown during the winter in unheated soil, or in artificially heated soil disinfected with methyl bromide, did not collapse. Root colonization by the pathogen was higher in the autumn and in heated soil than in the winter season in nonheated soil. Fifty per cent of root segments were colonized 35, 42 and 67 days after planting in the winter-heated, autumn and winter-unheated plots, respectively. A high correlation was found between soil temperatures above 20°C during the first 30 days after planting and disease severity. It is suggested that soil temperature during the early stages of plant development is an important factor in disease development and the expression of melon collapse caused by M. cannonballus.     

The effect of root-knot nematodes and Ethrel on Fusarium wilt of tomatoes

The reaction of two tomato varieties, Moneymaker (susceptible toFusarium) and Fortos (resistant toFusarium) to inoculation withMeloidogyne javanica, M. incognita andFusarium oxysporum f. sp.lycopersici race1 as well as to application of Ethrel (2 chloroethane phosphonic acid)—an ethylene releasing compound—was studied. With Moneymaker, Ethrel reduced wilt symptoms drastically and this was correlated with a reduction in the number of infected vascular bundles in the stem. Height of the stems was increased. Both nematode species counteracted the therapeutic effect of Ethrel by increasing the number of infected vascular bundles. Fortos remained resistant toFusarium in the presence of nematodes and/or Ethrel. Only, a slight tendency toward resistance-breaking by the nematode was observed. In this variety enhancement of the pathogenic effects of the nematodes by Ethrel was evidenced by increased gall size. Histological studies indicated no difference in xylem structure among the various treatments. These results support the evidence that plant growth regulators play a role in the pathogenicity of root-knot nematodes, theFusarium resistance of the plants, and the effect of nematodes on the severity ofFusarium wilt.Samenvatting Bestudeerd werd de reactie, van twee tomatecultivars, Moneymaker (gevoelig voorFusarium) en Fortos (resistent tegenFusarium) op inoculatie metMeloidogyne javanica, M. incognita enFusarium oxysporum f. sp.lycopersici ras 1, en op behandeling met Ethrel (2 chloorethaan fosfonzuur) een verbinding waaruit ethyleen vrijkomt. Bij Moneymaker resulteerde behandeling met Ethrel in een sterke afneming van verwelkingssymptomen gecorreleerd met een vermindering van metFusarium geinfecteerde vaatbundels in de stengel (Fig. 2 en 3) en een grotere lengte van de planten (Fig. 1). Beide aaltjessoorten hadden het omgekeerde effect en verhoogden het aantal metFusarium geïnfecteerde vaatbundels (Fig. 2 en 3).Bij Fortos was er slechts een zeer geringe mate van verlies van resistentie tegenFusarium onder invloed van de aanwezigheid van wortelknobbelaaltjes. Ethrel elimineerde de geringeFusarium infectie geheel. Bij deze cultivar werd het schadelijk effect van de aaltjes duidelijk versterkt door Ethrel in correlatie, met een sterke toeneming van de omvang der gallen (toeneming wortelgewicht, Fig. 4). Ethrel had geen rechtstreeks effect opFusarium in reincultures.Histologische waarnemingen gaven geen aanwijzingen voor het optreden van veranderingen in de structuur van het xyleem (thyllen) onder invloed, van de behandelingen.De resultaten ondersteunen de opvatting dat groeiregulatoren een rol spelen bij de pathogeniteit van wortelknobbelaaltjes, deFusarium-resistentie van de plant, en de invloed van aaltjes op de ontwikkeling van,Fusarium in de plant.     

Involvement of kaempferol in the defence response of virus infected Arabidopsis thaliana

The roles of several phenolic compounds in plant defence response have been extensively studied, yet little is known about the role of flavonoids in plant-virus interaction. Quantitative and qualitative changes of selected phenolics in Arabidopsis thaliana induced by Cucumber mosaic virus containing satellite RNA (CMVsat) infection were analysed accompanied by plant hormone, chalcone synthase and pathogenesis-related gene expression analysis. Lower leaves of infected plants had a lower concentration of total phenolics compared to control plants. The concentration of kaempferol in upper leaves of all infected plants was significantly lower compared to control plants, while the expression of the chalcone synthase gene in those leaves was in most cases upregulated. All infected plants had a higher concentration of indole-3-acetic acid in lower leaves, which was accompanied with a lower concentration of kaempferol in upper leaves. Our research demonstrates a correlation between kaempferol and indole-3-acetic acid in response to CMVsat infection in Arabidopsis. We demonstrated two different metabolic patterns in infected plants suggesting the activation of two different defence responses. We also propose kaempferol to be an important part of the auxin-dependent defence response which limits systemic movement of CMVsat and that this defence response is activated prior to the well-known salicylic acid dependent defence response. Further research on kaempferol and its role in Arabidopsis-CMVsat interaction will improve our understanding on the role of flavonoids in plant defence.     

Multilocus genes based characterization of phytoplasma strains associated with Mexican and French marigold species in India

European Journal of Plant Pathology - During the winters of 2018 to 2020, witches’ broom, phyllody, flat stem, little leaf, yellowing and stunting symptoms were recorded in&nbsp;Mexican...     

Kerala wilt disease phytoplasma: Phylogenetic analysis and identification of a vector, Proutista moesta

Kerala wilt disease of coconut palm is a major threat of coconut production in Kerala caused by phytoplasma. The genomic DNA purified from the insect tissues of Proutista moesta (PM) and Stephanitis typica (ST) was subjected to PCR assay using the primer combination P1/P6, P1/P7 and P4/P7. The amplified products resolved a prominent band of 650 bp for the universal primer P4/P7 and no bands were noticed for the primer pairs P1/P6 and P1/P7 combination. Since P4/P7 amplifies the 16S–23S intergenic spacer region of 16SrRNA gene, the PCR product 650 bp of the insect PM indicate the phytoplasma DNA. The presence of 650 bp for the primer P4/P7 in the genomic DNA isolated from P. moesta indicates the vectoral ability of the insect. No sign of amplification was noticed in the case of ST for the three sets of primers suggesting the inability of this insect as vector. The amplified product 650 bp from the genomic DNA of KWD palms as well as the insect tissues of P. moesta was gel purified and sequenced. The sequential similarity of 650 bp of both KWD phytoplasma and the insect phytoplasma supports the transmission of phytoplasma through the vector PM. Moreover, the sequence of 650 bp was compared with other sequences of 26 coconut phytoplasmas so far reported internationally and a cladogram was prepared for determining the phylogenetic status. It is obvious from the cladogram that the KWD disease phytoplasma is evolutionarily closest to coconut phytoplasma of coconut lethal yellowing of Mexican palms within the group 16SrIV. Phylogenetically, KWD phytoplasma is grouped in the new subgroup 16SrIV-C subsequent to the groups 16SrIV-A and 16SrIV-B for Mexican coconut lethal yellowing and Tanzanian coconut lethal decline, respectively. The restriction enzyme analysis of the PCR product 650 bp using the enzymes AluI, BclI, HindIII and RsaI further supports the phytoplasmic nature of DNA. This data records the first finding of the vector of Kerala wilt disease by detecting KWD phytoplasma in insect tissue of PM by PCR based methods. Moreover, the study reveals the phylogenetic status of KWD phytoplasma compared to other coconut phytoplasmas internationally.     

Detection and molecular characterization of phytoplasma associated with chickpea phyllody disease in south India

Chickpea (Cicer arietinum L.) plants showing typical symptoms of infection by a phytoplasma that causes phyllody disease have been commonly observed in recent years in parts of south India. The symptoms included pale green leaves, bushy appearance due to excessive stunting of shoots, reduced internodal length and excessive axillary proliferation. The causal agent of the phyllody disease was identified based on symptoms, amplification of 16S rDNA of the phytoplasma by polymerase chain reaction (PCR) from infected samples, as well as by sequencing and phylogenetic analysis. First round PCR and nested-PCR protocols were standardized for improved efficiency and reliability of the diagnostic protocols. Using the primers P1/P7 and R16F2n/R16R2, 1,800?bp and 1,200?bp size products were amplified in first round PCR and nested-PCR protocols, respectively. The PCR product was cloned and sequenced and compared with the reference phytoplasma sequences from the database (NCBI). The Indian chickpea phyllody phytoplasma 16S rDNA sequences shared the highest nucleotide identity (>98%) with the 16S rII group phytoplasma candidates, also infecting chickpea from Australia and Pakistan. This is the first report of a phytoplasma of the 16SrII-group infecting chickpea from India. The genetic similarities and the potential threat of this new disease to chickpea cultivation in India are discussed.     

Residues of methomyl in strawberries,tomatoes and cucumbers

An accurate and rapid high performance liquid chromatography method was developed to monitor residues of methomyl in plant extracts. The rate of disappearance of foliage-applied methomyl from strawberries, tomatoes and cucumbers was studied. Residues reached levels of 0.55, 0.2 and 0.6 mg kg^?1 seven days after methomyl had been applied to strawberries, tomatoes and cucumbers, respectively. Results also showed that rinsing treated fruits with tap water removed considerable amounts of methomyl. Samples of strawberries, tomatoes and cucumbers were collected from local markets at Ismailia, and checked for methomyl residues. Residues in 12.5% of tomato and 25% of strawberry samples were above 0.2 mg kg^?1.