Arbuscular mycorrhizal fungus inocula from coastal sand dunes arrest olive cutting growth under salinity stress.

Cultivation of olive trees covers large coastal areas of land in Mediterranean regions, many of them characterized by low soil fertility and exposed to salinity and seasonal drought. In this frame, we developed mixed community inocula of arbuscular mycorrhizal fungi (AMF) derived from the extreme, seasonally arid environments of six Mediterranean sand dunes and evaluated their effects, in the form of community inocula, on rooted semi-woody olive tree cuttings (Olea europaea cv. Koroneiki). The plantlets were grown in the greenhouse for 10 months under 50 mM and 100 mM concentrations of NaCl, successively applied to induce osmotic stress. Inoculation had a positive effect on plant growth and nutrient uptake. However, the three best-performing inocula in early colonization and in plant growth enhancement also resulted in high plant sensitivity to high salinity, which was not observed for the other three inocula. This was expressed by decreased nutrient uptake and drastically lower plant growth, plant photosynthesis, and stomatal conductance (generally an over 50% reduction compared to no salinity application). Amplicon sequencing analysis of the olive plants under salinity stress showed that the AMF communities in the roots were clearly differentiated by inoculation treatment. We could not, however, consistently associate the plant responses observed under high salinity with specific shared AMF community membership or assembly attributes. The observed physiological overreaction to osmotic stress may be an adaptation trait, potentially brought about by host selection coupled to abiotic environmental filtering, in the harsh conditions from which the AMF inocula were derived. The overreaction may, however, be undesirable if conveyed to allochthonous plants at an agronomic level.

First Report of Verticillium Wilt Caused by Verticillium dahliae on Avocado Trees in Greece.

Avocado (Persea americana) is an important crop for Chania, Crete, Greece, and is grown on more than 800 ha. In November 2013, 4-year-old trees in a new avocado grove of cv. Hass grafted onto the rootstock 'Bacon,' previously planted in citrus trees, showed symptoms of yellowing, leaf fall, twig and branch dieback and vascular tissue discoloration. Disease incidence was estimated at 2.3% (12 out of 530 trees affected). A fungus was consistently and readily isolated from symptomatic vascular tissue, previously surface-disinfested with 95% ethanol, on acidified potato dextrose agar (APDA). After 7 days, slow-growing colonies were transferred to PDA and the growth rate of the fungus was 2.9 mm/day at 24°C in the dark. Microscopic observations revealed hyaline hyphae with many irregular, dark microsclerotia measuring 40 to 200 × 30 to 75 µm (average 94.5 × 50.3 µm) developing after 21 days of growth. Hyaline, elliptical, single-celled conidia measuring 2.8 to 7.5 × 2.5 to 4.3 µm (average 4.8 × 3.1 µm) developed on verticillate conidiophores. For molecular characterization, Verticillium dahliae specific primer pair ITS1-F/ITS2-R that amplifies the rRNA internal transcribed spacer (ITS) region was used (2). Band of expected size was amplified, sequenced, and deposited in GenBank (Accession No. KJ818294). On the basis of morphological characteristics (3) and a BLAST search with 100% identity to the published ITS sequence of a V. dahliae isolate in GenBank (KC834733.1), the fungus was identified as V. dahliae. Five 1-year-old avocado plants of cv. Hass, grafted onto the rootstock 'Bacon,' were used for pathogenicity tests. Artificial inoculation was performed by making a 5.0 × 3.5 mm hole in the rootstock trunk, injecting approximately 40 µl of a 2.8 × 107 conidia/ml suspension into the vessels (spores were introduced passively), sealing with Vaseline, and covering with adhesive paper tape. Five control plants were mock inoculated with sterilized distilled water. Disease symptoms that appeared 18 days post artificial inoculation were similar to those observed under natural infection conditions. Thirty-five days post artificial inoculation, disease incidence was 80%, whereas the percentage of positive V. dahliae re-isolations from infected tissues was 95% (96.7 and 93.3% from rootstock and graft, respectively). The extent of vascular tissue discoloration from the point of inoculation ranged from 11 to 62 cm, whereas V. dahliae was successfully re-isolated even from the end of the graft (approximately 60 cm above the initial inoculation point), thus confirming Koch's postulates. Neither symptoms nor positive isolations were observed in control plants. The pathogenicity test was repeated twice with similar results. Verticillium wilt of avocado has been observed in several countries including Argentina, Chile, Ecuador, Israel, Mexico, Morocco, Spain, and the United States (1). To the best of our knowledge, this is the first report of Verticillium wilt on avocado in Greece. This disease could potentially be an increasing problem in areas where young avocado trees are established on land previously planted in vegetable crops. References: (1) J. C. Goud and J. A. Hiemstra. Chapter 3 in: A Compendium of Verticillium Wilt in Trees Species, 1998. (2) E. A. Markakis et al. Eur. J. Plant Pathol. 124:603, 2009. (3) G. F. Pegg and B. L. Brady. Verticillium Wilts. CABI Publishing, Wallingford, UK, 2002.

Strong host-specific selection and over-dominance characterize arbuscular mycorrhizal fungal root colonizers of coastal sand dune plants of the Mediterranean region.

Sand dunes of the Mediterranean region constitute drought-stressed, low-fertility ecosystems. Arbuscular mycorrhizal fungi (AMF) are regarded as key components of their biota, that contribute to plant host adaptation and fitness. However, AMF community assembly rules in the roots of the psammophilous plants of coastal sand dunes have not been investigated. We studied the root colonizing AMF communities of four characteristic native plants of eastern Mediterranean coastal foredunes, in nine locations in Greece. Host-specificity (plant identity) was the major driver of AMF community assembly in the plant roots, while geographical distance between locations was not related to differences in the AMF communities. Additionally, colonizer AMF communities were characterized by over-dominance of a single operational taxanomic unit (OTUs), which was remarkably host-specific among locations. Wider dissimilarity in AMF communities was observed in small and disturbed (SD) sites compared to large and undisturbed (LU) sites, a trait that may be attributed to relaxed environmental filtering and facilitated AMF dispersal/immigration in SD sites from surrounding habitats. Overall, our results indicate that the assembly of root-colonizing AMF communities in the eastern Mediterranean sand dunes is characterized by strong biotic filtering (host identity), suggesting that co-adaptation processes may be more pronounced than previously proposed, under extreme environmental conditions.

Carbon metabolism in developing soybean root nodules: the role of carbonic anhydrase.

A full-length cDNA clone encoding carbonic anhydrase (CA) was isolated from a soybean nodule cDNA library. In situ hybridization and immunolocalization were performed in order to assess the location of CA transcripts and protein in developing soybean nodules. CA transcripts and protein were present at high levels in all cell types of young nodules, whereas in mature nodules they were absent from the central tissue and were concentrated in cortical cells. The results suggested that, in the earlier stages of nodule development, CA might facilitate the recycling of CO2 while at later stages it may facilitate the diffusion of CO2 out of the nodule system. In parallel, sucrose metabolism was investigated by examination of the temporal and spatial transcript accumulation of sucrose synthase (SS) and phosphoenolpyruvate carboxylase (PEPC) genes, with in situ hybridization. In young nodules, high levels of SS gene transcripts were found in the central tissue as well as in the parenchymateous cells and the vascular bundles, while in mature nodules the levels of SS gene transcripts were much lower, with the majority of the transcripts located in the parenchyma and the pericycle cells of the vascular bundles. High levels of expression of PEPC gene transcripts were found in mature nodules, in almost all cell types, while in young nodules lower levels of transcripts were detected, with the majority of them located in parenchymateous cells as well as in the vascular bundles. These data suggest that breakdown of sucrose may take place in different sites during nodule development.

Lotus japonicus contains two distinct ENOD40 genes that are expressed in symbiotic, nonsymbiotic, and embryonic tissues.

ENOD40, an early nodulin gene, has been postulated to play a significant role in legume root nodule ontogenesis. We have isolated two distinct ENOD40 genes from Lotus japonicus. The transcribed regions of the two ENOD40 genes share 65% homology, while the two promoters showed no significant homology. Both transcripts encode a putative dodecapeptide similar to that identified in other legumes forming determinate nodules. Both ENOD40 genes are coordinately expressed following inoculation of roots with Mesorhizobium loti or treatment with purified Nod factors. In the former case, mRNA accumulation could be detected up to 10 days following inoculation while in the latter case the accumulation was transient. High levels of both ENOD40 gene transcripts were found in nonsymbiotic tissues such as stems, fully developed flowers, green seed pods, and hypocotyls. A relatively lower level of both transcripts was observed in leaves, roots, and cotyledons. In situ hybridization studies revealed that, in mature nodules, transcripts of both ENOD40 genes accumulate in the nodule vascular system; additionally, in young seed pods strong signal is observed in the ovule, particularly in the phloem and epithelium, as well as in globular stage embryos.