Isolation of Eutypa lata from Juglans regia, and Pathogenicity Studies on Different Wood Plants

The fungus Eutypa lata was isolated from diseased walnut trees (Juglans regia) exhibiting small cankers. The morphological characteristics of the culture and the pathogenicity were compared with those of known isolates of the fungus from other hosts. Inoculation tests with walnut isolates on grape, walnut, almond and apricot yielded characteristic cankers. Furthermore, pathogenicity tests on walnut with isolates from other different hosts resulted in differences in virulence. Similar differences in virulence were observed between 13 single ascospore isolates of the fungus ex apricot inoculated on walnut.     

Phenolic and heterocyclic metabolite profiles of the grapevine pathogen Eutypa lata

The ascomycete Eutypa lata is the causative agent of eutypa dieback in grapevines, a serious economic problem in major wine grape producing areas. In order to develop a predictive, non-destructive assay for early detection of fungal infection, the phenolic metabolite profiles of 11 strains of E. lata grown on four different artificial growth media were analyzed by HPLC and their variability compared with growth on Cabernet Sauvignon grapevine wood and wood extracts. Six compounds were generally produced in significant amounts, namely eutypinol, eulatachromene, and eutypine and its benzofuran cyclization product, together with siccayne and eulatinol. The two most widely distributed and abundant metabolites were eutypinol and eulatachromene, which were present in 8 of the strains grown on grapewood aqueous extract fortified with sucrose. Metabolite production on grapevine extract was greatly enhanced relative to the artificial media, indicating that this native substrate provides optimal conditions and a more representative profile of the metabolites produced in the natural disease state. The primary metabolites were tested in a grapeleaf disc bioassay to establish their relative toxicity. Neither eutypinol nor siccayne were phytotoxic; eulatachromene, eulatinol, eutypine, and the benzofuran exhibited necrotic effects in the bioassay. The results indicate that eutypa dieback may be caused by several E. lata metabolites rather than a single compound.     

Molecular Identification of Fomitiporia mediterranea and Eutypa lata/Libertella blepharis in Platanus × acerifolia

The studies of woody organs‐affecting diseases of Platanus × acerifolia can be hampered by the finding of fungi whose identification is difficult with mycological techniques. In a previous study on Platanus × acerifolia affected by severe cankers, Fomitiporia mediterranea/punctata‐like fungi, not associated with fruit bodies and Libertella sp. were recovered. Due to the severity of the associated symptoms, a characterization of fungal ribosomal DNA genes was undertaken in the present study, aimed to specific identification of the pathogens. From DNA of Fomitiporia‐vegetative isolates and Fomitiporia‐fruit body isolates, included in the study for comparison (from fruit body on plane trees typical of F. mediterranea/punctata), and Libertella sp., DNA fragments were amplified in polymerase chain reaction (PCR) with the use of ITS5/ITS4 and 5.8S‐R/LR7 primer pairs. Sequence alignments showed that Fomitiporia‐vegetative/fruit body isolates had highly homologous ITS5/ITS4 sequences, with a nucleotide identity of 98–100%. All the Fomitiporia isolates from plane tree showed 97–100% and 91–94% of nucleotide identity respectively with ITS5/ITS4 sequences of known strains of F. mediterranea and F. punctata, extracted from GenBank. The strong similarity of these identity ranges with those obtained within F. mediterranea (98–100%) and between the two Fomitiporia species (90–94%) confirms the identification of the isolates from plane tree as F. mediterranea. Sequence comparison between Libertella sp. from plane tree and known strains of Eutypa lata/L. blepharis showed 94–99% of nucleotide identity. The comparison with eight additional species of Eutypa showed 90–93% of nucleotide identity. As previously reported for the different taxa within Diatrypaceae, also ITS5/ITS4 sequence of Libertella sp. from plane tree exhibited 11‐bp tandem repeats motifs. Results of sequence alignments, of phylogenetic trees, and of the putative restriction map, identify the Fomitiporia isolates of this work as F. mediterranea, and the isolates of Libertella sp. as E. lata/L. blepharis. For comparative purposes, ITS5/ITS4 sequences of Spongipellis pachyodon and Fomes fomentarius from plane tree, were also obtained in this work.     

Inflorescence bud proteins of Pistacia vera

 The Pistacia vera L. (common name pistachio) is a unique dioecious and deciduous tree species, which is productive under harsh desert climates. We have identified and purified an Inflorescence Bud Protein of 32 kDa (IBP32) from male pistachio trees. There is a close correlation between its accumulation and inflorescence bud development and its disappearance and flowering. Using antibodies raised against this protein, we have identified in female trees the IBP32 and in addition a 27 kDa protein (IBP27), which appears to be specific to female inflorescence buds. The accumulation and disappearance of IBP27 follows the same pattern as that of IBP32. These proteins are glycoproteins rich in glycine and alanine and are highly hydrophilic. Based on the analytical results and immunological cross-reactivity between dehydrin antibodies and the IBPs, it is assumed that the latter are dehydrin-like and may protect inflorescence bud meristems against cold injury during dormancy. The IBPs are the major proteins of the pistachio bud, therefore they may also serve as nitrogen storage during winter for inflorescence bud growth in spring. Received: 17 October 1997 / Accepted: 6 March 1998     

Two new monoterpenes from the bled resin of Pistacia vera

Structure determination and synthesis of two novel bicyclic p-menthane monoterpenes isolated from Pistacia vera are reported.     

Antifungal effects of cysteine towards Eutypa lata, a pathogen of vineyards.

Cysteine inhibited mycelial growth of the pathogenic fungus affecting grapevines Eutypa lata Pers. Fr. Tul. and C. Tul. in a concentration-dependent manner. The threshold value (defined by the concentration inducing a growth inhibition higher than 5%) was 0.5 mM. A 10 mM concentration induced a complete inhibition of growth and triggered necrotic processes as evidenced by an increasing number of nuclei stained by propidium iodide. In conditions mimicking the plant environment (in particular, a pH near the apoplastic value, i.e. 5.5), 6 mM cysteine induced dramatic modifications in the structural organization of the mycelium (wall, mitochondria, vacuoles and nucleus) leading to death of the hyphae. The antifungal effect of the molecule increased at the acidic experimental pH (pH 4.1). The effect was highly specific to cysteine since modifying the molecular arrangement or masking the SH-function hindered the antifungal efficiency. Cysteine spectrum of action was broad among the various strains of E. lata tested. However, a lower efficiency was observed against fungal species intervening in other grapevine diseases (esca, black dead arm). Besides its direct antifungal effect, the role of cysteine presents particular interest in the fight against fungal pathogens since it triggered an excretion of ergosterol, a compound with elicitor properties. Therefore, cysteine may indirectly increase plant defense reactions.     

Identification of a RAPD marker linked to sex determination in Pistacia vera using bulked segregant analysis

The Random Amplified Polymorphic DNA (RAPD) technique was used to amplify DNA segments, with the objective of finding markers linked to sex determination in the dioecious species, Pistacia vera. Progenies from two female parents pollinated by a common male parent were studied. Two bulks of DNA were made in each cross, one from males and one from females, by pooling an equal weight of fresh leaves from each individual contributing to the bulk prior to DNA extraction. DNA was extracted from each bulked sample and from each of the contributing individuals. DNA was also extracted from 14 cultivars of P. vera and from 94 open-pollinated, fewweeks-old P. vera seedlings of unknown sex. Seven hundred different decamer oligonucleotide primers were used to perform DNA amplification, with 1 of these (OPO08) producing a 945 bp amplification band that was present only in the bulked female samples and absent in the bulked male samples of the two crosses. The relationship between band presence and female sex expression was conserved in every individual obtained from the two crosses and in the 14 cultivars unrelated to the crosses. We propose that this band is tightly linked to the gene(s) that control sex determination in pistachio. The OPO08₉₄₅ RAPD marker could be used in a breeding program to screen the gender of pistachio plants long before they reach reproductive maturity, resulting in considerable savings of time and economic resources. In order to verify that assumption we screened 94 additional seedlings with the OPO08 primer and obtained results consistent with a 11 male:female ratio.     

Germination of Pistacia vera L. pollen in liquid medium

Summary The osmotic effect of Polyethylene glycol (PEG) has been shown to be sufficient to induce the germination of Pistacia vera L. pollen in liquid medium. The prehydration of the pollen in a saturated atmosphere for approximately 10 h was necessary to obtain maximum in vitro germination. Imbibition of the pollen in water resulted in the rapid leakage of solutes into the medium. These solutes consisted of approximately 50% carbohydrates, of which sucrose (0.65 mol/mg), glucose (0.77 mol/mg) and fructose (0.78 mol/mg) were the major sugars; the remaining 50% comprised proteins with the following major molecular weights 63 kDa, 60 kDa, 59 kDa, 40 kDa, 36 kDa, 35.5 kDa, 31 kDa, other organic matter and minerals.     

阿月浑子与中国黄连木叶形态结构特征研究

采用指甲油印迹法、石蜡切片观察、电镜观察的方法对阿月浑子与中国黄连木叶片解剖结构、气孔分布与形态特征进行观测分析。结果显示,阿月浑子叶上表皮气孔分布密度为132～P168个/cm2,且气孔多出现在主脉附近,下表皮气孔数从基部到叶尖逐渐减少,密度为112～P357个/cm2;中国黄连木上表皮气孔极少,仅出现在主脉处,下表皮气孔分布状况与阿月浑子相近,密度为164～P377个/cm2;两树种均分布有巨型气孔和气孔群、表皮细胞角质层发达、少量非腺毛主要分布在叶缘,其次在主脉处,叶面有蜡质分布,而且阿月浑子的蜡质明显比中国黄连木多;阿月浑子有4～P5层栅栏组织细胞,几乎没有海绵组织,中国黄连木叶肉具1层栅栏组织,多层海绵组织;阿月浑子主叶脉6～P8个维管束环状排列,在韧皮部中有6～P8个内分泌道,中国黄连木主叶脉3个维管束扇形排列,在韧皮部中有1～P3个内分泌道。对2树种叶形态结构特征与抗旱性的关系进行分析结果表明,阿月浑子具有旱生植物叶片结构特点,其水分利用方式属耗水型,对我国北方气候表现出一定的适应性。     

The in vitro effects of selected substances and nanomaterials against Diaporthe eres,Diplodia seriata and Eutypa lata

Grapevine trunk pathogens (GTPs) cause serious damage to grapevines and have significant economic impacts. There is no effective protection against grapevine trunk diseases. Newly designed AgSe nanoparticles (NPs) and CuSe NPs, single-element Ag NPs, Cu NPs, Se NPs and selected chemicals or chemical agents such as sodium arsenite, 8-hydroxyquinoline, silver nitrate, colloidal silver, Altron Silver fertilizer and silver thiosulfate complex (NH₄)₃/Ag(S₂O₃)₂ were tested in vitro against two serious GTPs Diaporthe eres, Eutypa lata and Diplodia seriata isolated from walnut. The most significant inhibition of fungal growth was observed for silver nitrate and AgSe NPs, which showed the highest level of half the maximum effective EC₅₀ concentration with the lowest concentrations. In the case of silver nitrate at a concentration of 1000 mg L⁻¹, 79% inhibition of mycelial growth was observed for the pathogen E. lata, 48% for D. seriata and 54% for D. eres. AgSe NPs, in which the concentration of silver is 2588 mg L⁻¹ and that of selenium is 902 mg L⁻¹, showed 68% inhibition of mycelial growth in the pathogen E. lata, 54% in D. seriata and 58% in D. eres.     

Pistillate and staminate flower development in dioecious Pistacia vera (Anacardiaceae)

The development of staminate and pistillate flowers in the dioecious tree species Pistacia vera L. (Anacardiaceae) was studied by scanning electron microscopy with the objective of determining organogenetic patterns and phenology of floral differentiation. Flower primordia are initiated similarly in trees of both sexes. Stamen and carpel primordia are initiated in both male and female flowers, and the phenology of organ initiation is essentially identical for flowers of both sexes. Vestigial stamen primordia arise at the flanks of pistillate flower apices at the same time functional stamens are initiated in the staminate flowers. Similarly, a vestigial carpel is initiated in staminate flowers at the same time the primary, functional carpel is initiated in pistillate flower primordia. Differences between the two sexes become apparent early in development as, in both cases, development of organs of the opposite sex becomes arrested at the primordial stage. Male flowers produce between four and six mature functional stamens and female flowers produce a gynoecium with one functional and two sterile carpels.     

Plant regeneration from encapsulated embryoids and an embryogenic mass of pistachio, Pistacia vera L.

Pieces of an embryogenic mass (EMS) induced in culture from immature fruits of pistachio, Pistacia vera L., were encapsulated into calcium alginate beads. Somatic embryos were also encapsulated individually into calcium alginate beads to produce synthetic seeds. The viability of the encapsulated EMS and somatic embryos was investigated immediately following encapsulation, and after storage for 60 days at 4°C. The encapsulated-stored EMS fragments recovered their original proliferative capacity after two months storage following two sub-cultures, but non-encapsulated-stored EMS failed to recover. The conversion frequency of synthetic seeds to seedling plants was 14% after storage for 60 days at 4°C, from which it may be concluded that encapsulation is a practical procedure for short-term storage of embryogenic pistachio tissue, and may be applicable to the preservation of desirable elite genotypes.Abbreviations BAP Benzylaminopurine - EMS(es) Embryogenic mass(es) - MS Murashige and Skoog medium (Sigma M-0404) - PGR(s) Plant growth regulator(s)     

Control of anthocyanin biosynthesis pathway gene expression by eutypine,a toxin from Eutypa lata,in grape cell tissue cultures

Eutypine, 4-hydroxy-3-(3-methyl-3-butene-1-ynyl) benzaldehyde, is a toxin produced by Eutypa lata, the causal agent of Eutypa dieback in grapevine. The effect of the toxin on anthocyanin synthesis has been investigated in Vitis vinifera cv. Gamay cell cultures. At concentrations higher than 200 micromol/L, eutypine reduced anthocyanin accumulation in cells. The reduction in anthocyanin accumulation was proportional to the eutypine concentrations and HPLC analysis showed that eutypine affected the levels of all anthocyanins. The effect of eutypine application on the expression of five genes of the anthocyanin biosynthesis pathway, including chalcone synthase (CHS), flavonone-3-hydroxylase (F3H), dihydroflavonol 4-reductase (DFR), leucoanthocyanidin dioxygenase (LDOX), and UDP glucose-flavonoid 3-O-glucosyl transferase (UFGT) was determined. Expression of CHS, F3H, DFR and LDOXwas not affected by the addition of eutypine to grapevine cell cultures. In contrast, expression of the UFGT gene was dramatically inhibited by the toxin. These results suggest that in grapevine cell cultures, eutypine strongly affects anthocyanin accumulation by inhibiting UFGT gene expression. The mechanism of action of eutypine is discussed.     

Influence of temperature and nutritional requirements for mycelial growth of Eutypa lata, a vineyard pathogenic fungus

Eutypa dieback (dying arm disease, eutypiosis) is a very devastating disease in many grape-producing areas around the world. This vascular disease is induced by the ascomycete Eutypa lata Pers. Fr. Tul & C. Tul. invading the trunk by pruning wounds. The environmental factors and the nutritional requirements regulating fungus development are yet poorly known. This work shows that the isolated strain of E. lata was able to grow in a large temperature range (2-30 degrees C). However, a higher temperature (35 degrees C) presented inhibitory effects on mycelial growth. E. lata was able to use various osidic molecules (C5, C6, C12, C18, C24, and starch); showing thus a large adaptation to the carbon source supplied. As nitrogen source, it used salts and numerous natural amino acids. A significant result was obtained with cysteine presenting obvious antifungal properties. This effect can further be used with the aim of setting up a curative treatment of the disease.     

Identification of sex-linked SNP markers using RAD sequencing suggests ZW/ZZ sex determination in Pistacia vera L.

Background

Pistachio (Pistacia vera L.) is a dioecious species that has a long juvenility period. Therefore, development of marker-assisted selection (MAS) techniques would greatly facilitate pistachio cultivar-breeding programs. The sex determination mechanism is presently unknown in pistachio. The generation of sex-linked markers is likely to reduce time, labor, and costs associated with breeding programs, and will help to clarify the sex determination system in pistachio.

Results

Restriction site-associated DNA (RAD) markers were used to identify sex-linked markers and to elucidate the sex determination system in pistachio. Eight male and eight female F₁ progenies from a Pistacia vera L. Siirt × Bağyolu cross, along with the parents, were subjected to RAD sequencing in two lanes of a Hi-Seq 2000 sequencing platform. This generated 449 million reads, comprising approximately 37.7 Gb of sequences. There were 33,757 polymorphic single nucleotide polymorphism (SNP) loci between the parents. Thirty-eight of these, from 28 RAD reads, were detected as putative sex-associated loci in pistachio. Validation was performed by SNaPshot analysis in 42 mature F₁ progenies and in 124 cultivars and genotypes in a germplasm collection. Eight loci could distinguish sex with 100% accuracy in pistachio. To ascertain cost-effective application of markers in a breeding program, high-resolution melting (HRM) analysis was performed; four markers were found to perfectly separate sexes in pistachio. Because of the female heterogamety in all candidate SNP loci, we report for the first time that pistachio has a ZZ/ZW sex determination system. As the reported female-to-male segregation ratio is 1:1 in all known segregating populations and there is no previous report of super-female genotypes or female heteromorphic chromosomes in pistachio, it appears that the WW genotype is not viable.

Conclusion

Sex-linked SNP markers were identified and validated in a large germplasm and proved their suitability for MAS in pistachio. HRM analysis successfully validated the sex-linked markers for MAS. For the first time in dioecious pistachio, a female heterogamety ZW/ZZ sex determination system is suggested.