Soledad Martos

First Report of Lasiodiplodia theobromae Associated with Decline of Grapevine Rootstock Mother Plants in Spain

A field of Richter 110 rootstock mother plants in Valencia Province (eastern Spain) was surveyed during November 2006 to study the mycoflora of declining plants. Two canes with stunted leaves were collected from a plant with a reduced number of shoots. No cankers or vascular lesions were observed in the collected canes. Six wood chips (1 to 2 mm thick) were taken from one basal fragment (3 to 4 cm long) of each cane, surface sterilized in 70% ethanol for 1 min, and plated on malt extract agar supplemented with 0.5 g L-1 of streptomycin sulfate. Petri dishes were incubated for 7 days at 25°C. A fungus was consistently isolated from all samples that showed the following characteristics: colonies grown on potato dextrose agar (PDA) at 25°C developed a white, aerial mycelium that turned gray after 4 to 6 days and produced pycnidia after 1 month on sterile grapevine slivers of twigs placed on the PDA surface; conidia from culture were ellipsoidal, thick walled, initially hyaline, nonseptate, and measuring 20 to 25 (22.5) × 12 to 14 (13) μm; aged conidia were brown, 1-septate with longitudinal striations in the wall; and pseudoparaphyses variable in form and length were interspersed within the fertile tissue. The fungus was identified as Lasiodiplodia theobromae (Pat.) Griffon & Maubl. from the above characteristics (2). Identity was confirmed by analysis of the nucleotide sequences of the internal transcribed spacer (ITS) region from the rRNA repeat and part of the translation elongation factor 1-alpha (EF1-α) and the β-tubulin (B-tub) genes, as done elsewhere (1,3). BLAST searches at GenBank showed a high identity with reference sequences (ITS: 100%, EF1-α: 97%; B-tub: 99%). Representative sequences of the studied DNA regions were deposited at GenBank (Accession Nos.: ITS: EU254718; EF1-α: EU254719; and B-tub: EU254720). A pathogenicity test was conducted on 1-year-old grapevine plants cv. Macabeo grafted onto Richter 110 rootstocks maintained in a greenhouse. A superficial wound was made on the bark of 10 plants with a sterilized scalpel, ≈10 cm above the graft union. A mycelial plug obtained from the margin of an actively growing fungal colony (isolate JL664) was placed in the wound and the wound was wrapped with Parafilm. Ten additional control plants were inoculated with sterile PDA plugs. All control plants grew normally, and the inoculation wound healed 3 months after inoculation. Plants inoculated with L. theobromae showed no foliar symptoms in the same period, but developed cankers variable in size surrounding the inoculation sites. Vascular necroses measuring 8.4 ± 1.5 cm (mean ± standard error) developed in the inoculated plants that were significantly longer than the controls (0.3 ± 0.2 cm). The pathogen was reisolated from all inoculated plants and no fungus was reisolated from the controls. These results confirmed the pathogenicity of L. theobromae to grapevine and points to a possible involvement of L. theobromae in the aetiology of grapevine decline as previously reported (3,4). To our knowledge, this is the first report of L. theobromae isolated from grapevine in Spain. References: (1) J. Luque et al. Mycologia 97:1111, 2005. (2) E. Punithalingam. No. 519 in: Descriptions of Pathogenic Fungi and Bacteria. CMI, Kew, Surrey, UK, 1976. (3) J. R. Úrbez-Torres et al. Plant Dis. 90:1490, 2006. (4) J. M. van Niekerk et al. Phytopathol. Mediterr. 45(suppl.):S43, 2006.

First Report of Canker Disease Caused by Neofusicoccum australe on Eucalyptus and Pistachio in Spain

In 2005 and 2006, dieback and branch cankers were observed in 12-year-old Eucalyptus globulus Labill. plantations in Gijón (northern Spain) and a 20-year-old pistachio (Pistacia vera L.) plantation in Constantí (northeastern Spain). Isolations were made from symptomatic branches. Small pieces of necrotic tissues were surface sterilized for 1 min in 1.5% NaOCl and plated onto malt extract agar amended with 0.5 g L-1 streptomycin sulfate. Plates were incubated at 25°C in the dark and all growing colonies were transferred to potato dextrose agar (PDA). A Neofusicoccum sp. was consistently isolated from necrotic tissues of both host species. On PDA at 25°C, isolates developed a moderately dense mycelium, initially with a pale yellow pigment diffusing into the medium but becoming olivaceous gray after 5 to 6 days. Pycnidia were produced on sterile eucalyptus and pistachio twigs placed on the surface of water agar after 1 month. Conidia were hyaline, fusiform, aseptate, with granular contents. Conidia from eucalyptus isolates measured (22.5-) 25.4 (-28.1) × (5-) 6.2 (-7.5) μm, (n = 40) and (20.0-) 23.6 (-28.0) × (6.5-) 7.1 (-8.0) μm, (n = 40) from pistachio isolates. Isolates were identified as Neofusicoccum australe (Slippers, Crous & M.J. Wingf.) Crous, Slippers & A.J.L. Phillips (1,2). DNA sequences of the rDNA internal transcribed spacer region (ITS), part of the beta-tubulin (BT2), and part of the translation elongation factor 1-alpha (EF1-α) genes from isolates CBS 122027 (pistachio) and CBS 122026 and CBS 122025 (eucalyptus) were used to confirm the identifications through BLAST searches in GenBank. Representative sequences of all studied regions were deposited in GenBank (ITS: EU375516 and EU375517; BT2: EU375520; EF1-α: EU375518 and EU375519). Pathogenicity tests were conducted on 8-month-old eucalyptus seedlings and 2-year-old pistachio plants with the three N. australe strains mentioned above. A mycelial plug taken from the margin of an actively growing colony of each isolate was put in a shallow wound (0.4 cm2) made with a scalpel on the stem of each plant. Inoculation wounds were wrapped with Parafilm. Controls were inoculated with sterile PDA plugs. Ten replicates for each isolate and plant species were used, with an equal number of control plants. Plants were maintained in a greenhouse at 25°C. After 3 weeks, all eucalyptus seedlings showed leaf wilting, stem canker, and pycnidia formation around the inoculation site. No foliar symptoms were observed in pistachio plants after 3 months, but depressed cankers variable in size and pycnidia formation developed around the inoculation site. Vascular necroses that developed on the inoculated plants were 10.2 ± 1.2 cm long in eucalyptus and 6.4 ± 1.6 cm long in pistachio, significantly greater than their respective controls (P < 0.01). There were no significant differences in necrosis lengths among the three N. australe isolate inoculations, irrespective of the inoculated host. These results point to a high susceptibility of eucalyptus to N. australe. No symptoms were visible in the control seedlings and no fungus was isolated from them. The pathogen was reisolated from all inoculated plants. To our knowledge, this is the first report of N. australe causing canker disease on eucalyptus and pistachio trees in Spain. References: (1) P. Crous et al. Stud. Mycol. 55:235, 2006. (2) B. Slippers et al. Mycologia 96:1030, 2004.

Characterization of Zinc and Cadmium Hyperaccumulation in Three Noccaea (Brassicaceae) Populations from Non-metalliferous Sites in the Eastern Pyrenees

The Southern slope of the Pyrenees is the meridional limit for the distribution of several Noccaea populations. However, the systematic description of these populations and their hyperaccumulation mechanisms are not well established. Morphological and genetic analysis (ITS and 3 chloroplast regions) were used to identify Noccaea populations localized on non-metallicolous soils during a survey in the Catalonian Pyrenees. Cd and Zn concentrations were analyzed in soils and plants both sampled in the field and grown hydroponically. The expression of selected metal transporter genes was assessed by quantitative PCR. The populations were identified as Noccaea brachypetala (Jord.) F.K. Mey by conspicuous morphological traits. Principal component analysis provided a clear separation among N. brachypetala, Noccaea caerulescens J. Presl & C. Presl and Noccaea occitanica (Jord.) F.K. Mey., three Noccaea species reported in the Pyrenees. Contrastingly, ITS and cpDNA analyses were unable to clearly differentiate these taxa. Differences in the expression of the metal transporter genes HMA3, HMA4, and MTP1 between N. caerulescens and N. brachypetala, and those amongst the N. brachypetala populations suggest differences in the strategies for handling enhanced Cd and Zn availability. This is the first report demonstrating Cd and Zn hyperaccumulation by N. brachypetala both in the field and in hydroponics. This comprehensive study based on taxonomic, molecular, and physiological data allows both the correct identification of this species and the characterization of population differences in hyperaccumulation and tolerance of Zn and Cd.

Zinc triggers signaling mechanisms and defense responses promoting resistance to Alternaria brassicicola in Arabidopsis thaliana.

According to the elemental defense hypothesis the accumulation of trace elements by plants may substitute for organic defenses, while the joint effects hypothesis proposes that trace elements and organic defenses can have additive or synergistic effects against pathogens or herbivores. To evaluate these hypotheses the response of the pathosystem Alternaria brassicicola-Arabidopsis thaliana to control (2μM) and surplus (12μM) Zn was evaluated using the camalexin deficient mutant pad3-1 and mtp1-1, a mutant with impaired Zn vacuolar storage, along with the corresponding wildtypes. In vitro, a 50% inhibition of fungal growth was achieved by 440μM Zn. A. thaliana leaves could accumulate equivalent concentrations without harm. In fact, surplus Zn enhanced the resistance of A. thaliana to fungal attack in Columbia (Col-0), Wassilewskija (WS), and mtp1-1. However, surplus Zn was unable to protect pad3-1 demonstrating that Zn cannot substitute for camalexin, the main organic defense in A. thaliana. High, non phytotoxic leaf Zn concentrations enhanced the resistance to A. brassicicola of A. thaliana genotypes able to produce camalexin. This was mainly due to Zn-induced enhancement of the JA/ETH signaling pathway leading to enhanced PAD3 expression. These results support the joint effects hypothesis and highlight the importance of adequate Zn supply for reinforced pathogen resistance.

Zinc hyperaccumulation substitutes for defense failures beyond salicylate and jasmonate signaling pathways of Alternaria brassicicola attack in Noccaea caerulescens

The hypothesis of metal defense as a substitute for a defective biotic stress signaling system in metal hyperaccumulators was tested using the pathosystem Alternaria brassicicola-Noccaea caerulescens under low (2 µM), medium (12 µM) and high (102 µM) Zn supply. Regardless the Zn supply, N. caerulescens responded to fungal attack with the activation of both HMA4 coding for a Zn transporter, and biotic stress signaling pathways. Salicylate, jasmonate, abscisic acid and indoleacetic acid concentrations, as well as biotic stress marker genes (PDF1.2, CHIB, LOX2, PR1 and BGL2) were activated 24 h upon inoculation. Based on the activation of defense genes 24 h after the inoculation an incompatible fungal-plant interaction could be predicted. Nonetheless, in the longer term (7 days) no effective protection against A. brassicicola was achieved in plants exposed to low and medium Zn supply. After 1 week the biotic stress markers were even further increased in these plants, and this compatible interaction was apparently not caused by a failure in the signaling of the fungal attack, but due to the lack of specificity in the type of the activated defense mechanisms. Only plants receiving high Zn exhibited an incompatible fungal interaction. High Zn accumulation in these plants, possibly in cooperation with high glucosinolate concentrations, substituted for the ineffective defense system and the interaction turned into incompatible. In a threshold-type response, these joint effects efficiently hampered fungal spread and, consequently decreased the biotic stress signaling.