Detection of Cherry Leaf Roll Virus in intensively managed grafted English (Persian) walnut trees in Italy

Blackline disease, caused by Cherry leaf roll virus (CLRV), is considered a serious threat limiting English walnut (Juglans regia L.) production in Italy and the EU if walnut species other than J. regia are used as rootstock. In spring 2014, canopy decline or death of several walnut trees associated with presence of a necrotic strip at the rootstock-scion junction was observed on plants grafted onto \u2018Paradox' (J. hindsii 7 J. regia) in a commercial orchard located in the Veneto region (north-eastern Italy). To ascertain the presence of CLRV in this orchard and in other walnut intensively managed orchards located in the same region, a monitoring was carried out in 2014- 2015

Occurrence and Etiology of Brown Apical Necrosis on Persian (English) Walnut Fruit.

In 1998, a severe fruit drop was observed in Italy, principally on cv. Lara Persian (English) walnut (Juglans regia). Dropped fruit showed a brown patch at the blossom end and blackening and rot of inner tissues. The disease, called brown apical necrosis (BAN), was investigated on fruit collected in Italy and France in 1999. In 2000, studies were carried out in three walnut orchards located in Italy and in France to substantiate the etiology of BAN. Isolations performed from inner diseased fruit tissues yielded several fungi, in decreasing frequency of isolation: species of Fusarium and Alternaria, and one species each of Cladosporium, Colletotrichum, and Phomopsis. However, only Fusarium spp. were recovered from stigmas of BAN-affected fruit. The fungi associated with BAN-diseased fruit and species composition differed among locations and over time, confirming results obtained in previous investigations. The species of Fusarium used in pathogenicity tests reproduced BAN-disease symptoms when inoculated on fruit, whereas an Alternaria alternata isolate caused only limited necrosis of the style. However, the role of the other fungi commonly isolated from BAN-diseased fruit remains to be defined. The walnut blight pathogen, Xanthomonas arboricola pv. juglandis, occasionally was isolated from BAN-diseased fruit. No correlation was found between the extent of external brown patches and the size of inner lesions. Repeated isolations from and inoculations of fruit demonstrated that BAN can be considered a complex disease, and the inner infections originate from the style of the fruit

Distinct colonization patterns and cDNA-AFLP transcriptome profiles in compatible and incompatible interactions between melon and different races of Fusarium oxysporum f. sp. melonis

Background: Fusarium oxysporum f. sp. melonis Snyd. & Hans. (FOM) causes Fusarium wilt, the most important infectious disease of melon (Cucumis melo L.). The four known races of this pathogen can be distinguished only by infection on appropriate cultivars. No molecular tools are available that can discriminate among the races, and the molecular basis of compatibility and disease progression are poorly understood. Resistance to races 1 and 2 is controlled by a single dominant gene, whereas only partial polygenic resistance to race 1,2 has been described. We carried out a large-scale cDNA-AFLP analysis to identify host genes potentially related to resistance and susceptibility as well as fungal genes associated with the infection process. At the same time, a systematic reisolation procedure on infected stems allowed us to monitor fungal colonization in compatible and incompatible host-pathogen combinations. Results: Melon plants (cv. Charentais Fom-2), which are susceptible to race 1,2 and resistant to race 1, were artificially infected with a race 1 strain of FOM or one of two race 1,2 w strains. Host colonization of stems was assessed at 1, 2, 4, 8, 14, 16, 18 and 21 days post inoculation (dpi), and the fungus was reisolated from infected plants. Markedly different colonization patterns were observed in compatible and incompatible host-pathogen combinations. Five time points from the symptomless early stage (2 dpi) to obvious wilting symptoms (21 dpi) were considered for cDNA-AFLP analysis. After successful sequencing of 627 transcript-derived fragments (TDFs) differentially expressed in infected plants, homology searching retrieved 305 melon transcripts, 195 FOM transcripts expressed in planta and 127 orphan TDFs. RNA samples from FOM colonies of the three strains grown in vitro were also included in the analysis to facilitate the detection of in planta-specific transcripts and to identify TDFs differentially expressed among races/strains. Conclusion: Our data suggest that resistance against FOM in melon involves only limited transcriptional changes, and that wilting symptoms could derive, at least partially, from an active plant response. We discuss the pathogen-derived transcripts expressed in planta during the infection process and potentially related to virulence functions, as well as transcripts that are differentially expressed between the two FOM races grown in vitro. These transcripts provide candidate sequences that can be further tested for their ability to distinguish between races. Sequence data from this article have been deposited in GenBank, Accession Numbers: HO867279-HO867981

Environmental and Pathogenic Factors Inducing Brown Apical Necrosis on Fruit of English (Persian) Walnut

Brown apical necrosis (BAN) is a most recently described disease affecting English (Persian) walnut fruit. BAN was only recorded in intensively managed walnut orchards and was found to be a disease complex mainly caused by Fusarium species. All fungi associated with this disease are polyphagous and ubiquitous, not specific to walnut. Consequently, BAN occurrence is more strictly dependent, than generally, on the interaction between pathological features and environmental conditions. Environmental variables identified with regression analysis showed that maximum temperature, angle of main wind direction versus tree row orientation, and orchard distance to the closest river/canal, all representative of climatic conditions occurring in the orchard, were related to fruit drop. The factor displaying the highest influence on severity of BAN fruit drop was maximum temperature and only subordinately factors are associated with relative humidity. BAN symptoms were reproduced with in planta artificial inoculation, and fruit drop of symptomatic fruit was significantly higher than that of the noninoculated trees for each type of inoculum (Fusarium semitectum, F. graminearum, and Alternaria spp.). F. semitectum and F. graminearum were more aggressive than Alternaria species, and the earliest artificial inoculations in mid-May resulted in the highest fruit drop. The extension of walnut fruit susceptibility and the conducive environmental factors to BAN are discussed

Reference gene selection for gene expression analysis in melon infected by Fusarium oxysporum f.sp. melonis.

Eleven candidate reference genes were evaluated in stem tissues of two melon (Cucumis melo L) genotypes infected with Fusarium oxysporum f.sp. melonis (FOM) race 1, to investigate the most stable internal control gene to use in gene expression study in melon under biotic stress. The candidate reference genes, utilized in the qRT-PCR, were selected among the most common reference genes reported in melon literature as well as on melon sequences found in available databases. The expression stability of the reference genes selected, was tested by using the geNorm and NormFinder statistical algorithms. Irrespective of the plant response to FOM infection, there was substantial agreement in gene ranks of both software. The ribosomal protein L2 gene had the high transcript stability while the elongation factor, cyclophilin7 and 18S rRNA were the least stable. The best three reference genes, ribosomal protein L2, actin* and cyclophilin were used to normalize the expression data of the glutathione S-transferase in the resistant and susceptible plants. Differences in the expression data emerged for both genotypes and were more evident in the resistant one. The ribosomal protein L2 showed similar normalized expression values in both genotypes and thus it could be indicated as unique internal control for gene expression analysis in melon under FOM infection. This work represents the first in-depth study on reference genes validation in melon under biotic stress condition. The identification of such genes would improve the accuracy and reliability of gene expression studies and speed up candidate disease resistant gene analysis in melon

TaqMan qPCR Detection and Quantification of <i>Phytophthora cinnamomi</i> in Soil and Plant Tissues for Walnut Disease Management

Phytophthora cinnamomi is a devastating soil-borne plant pathogen. The primary source of P. cinnamomi infection is the soil, where the pathogen can persist for long periods. Effective prevention and management of this pathogen in tree crops requires an early and reliable detection method. In this study, we developed a simple, fast, reliable, and sensitive method based on real-time quantitative PCR (qPCR) for P. cinnamomi detection and quantification directly in plant or soil samples. Primers were developed targeting the nuclear single-copy ras-related protein gene Ypt1, suitable for Phytophthora-specific PCR. The specificity of the assay was confirmed by testing it against genomic DNA from 50 isolates across eight different Phytophthora clades, including the very similar P. parvispora. The efficiency and reliability of the qPCR protocol were evaluated in challenging environmental samples, such as plant tissue of different host trees (walnut, chestnut, oak) and naturally infected soils in walnut orchards. The main outcome was the development of a qPCR method for the specific identification and quantification of P. cinnamomi in natural soil samples. Additionally, this study established a systematic and repeatable soil sampling method and developed an efficient soil DNA extraction technique to apply the developed qPCR in naturally infested soils of walnut orchards

Pro-apoptotic and cytostatic activity of naturally occurring cardenolides

Cardenoliddes are steroid glycosides which are known to exert cardiotonic effects by inhibiting the Na+/K+-ATPase. Several of these compds. have been shown also to possess anti-tumor potential. The aim of the present work was the characterization of the tumor cell growth inhibition activity of four cardenolides, isolated from Periploca graeca L., and the mechanisms underlying such an effect. The pro-apoptotic and cytostatic effect of the compds. was tested in U937 (monocytic leukemia) and PC3 (prostate adenocarcinoma). Characterization of apoptosis and cell cycle impairment was obtained by cytofluorimetry and WB. Periplocymarin and periplocin were the most active compds., periplocymarin being more effective than the ref. compd. ouabain. The redn. of cell no. by these two cardenolides was due in PC3 cells mainly to the activation of caspase-dependent apoptotic pathways, while in U937 cells to the induction of cell cycle impairment without extensive cell death. Interestingly, periplocymarin, at cytostatic but non-cytotoxic doses, was shown to sensitize U937 cells to TRAIL. Taken together, our data outline that cardiac glycosides are promising anticancer drugs and contribute to the identification of new natural cardiac glycosides to obtain chem. modified non-cardioactive/low toxic derivs. with enhanced anticancer potency

Thioredoxin system modulation by plant and fungal secondary metabolites

Thioredoxin (Trx) is the major cellular protein disulfide reductase in a broad range of organisms, including humans. Trx, together with glutaredoxin (Grx), plays critical roles in the regulation of cellular protein redox homeostasis. Reduced thioredoxin transfers reducing equivalents to disulphides in target proteins, leading to reversible oxidation of its active centre dithiol to a disulphide. The resulting disulphide bridge is, in turn, reduced to a dithiol by thioredoxin reductase (TrxR). Increasing attention has been paid to the role of Trx, as it has been shown to be a signalling intermediate beyond its intrinsic antioxidant activity. Indeed, this protein acts as a growth factor, activates a number of transcription factors regulating cell growth and survival, acts as cofactor for ribonucleotide reductase, and promotes angiogenesis. In addition, Trx have been demonstrated to cooperatively inhibit programmed cell death. Because of the multiple roles of Trx system in tumorigenesis, this protein represents an emerging target for anti-cancer drugs. Several Trx system modulators have been identified: a semi-synthetic Trx inhibitor, PX-12 (1-methylpropyl 2-imidazolyl disulfide), has been placed in a clinical trial. However, there is a growing interest in finding new selective ones. Natural products continue to provide structurally complex, but highly original lead structures for drug discovery programs: polyphenols, quinones, and terpenoids showed to affect the Trx/TrxR system at different levels. The purpose of this review is to provide an overview of the plant and fungal secondary metabolites interfering with Trx and TrxR activities, paying particularly attention on their mechanism of action. Among polyphenols, curcumin and some flavonoids such as myricetin and quercetin, have been identified as potential anticancer agents with a mechanism of action that may be mediated by the Trx system