Drought responsiveness in six wheat genotypes: identification of stress resistance indicators.

Introduction: Wheat (Triticum aestivum L.) is among the world's most important staple food crops. In the current climate change scenario, a better understanding of wheat response mechanisms to water stress could help to enhance its productivity in arid ecosystems. Methods: In this study, water relations, gas exchange, membrane integrity, agronomic traits and molecular analysis were evaluated in six wheat genotypes (D117, Syndiouk, Tunisian durum7 (Td7), Utique, Mahmoudi AG3 and BT) subjected to drought-stress. Results and discussion: For all the studied genotypes, drought stress altered leaf area, chlorophyll content, stomatal density, photosynthetic rate and water-use efficiency, while the relative water content at turgor loss point (RWC0) remained stable. Changes in osmotic potential at turgor loss point (Ψπ0), bulk modulus of elasticity (Ɛmax) and stomatal regulation, differed greatly among the studied genotypes. For the drought-sensitive genotypes AG3 and BT, no significant changes were observed in Ψπ0, whereas the stomatal conductance (gs) and transpiration rate (E) decreased under stress conditions. These two varieties avoided turgor loss during drought treatment through an accurate stomatal control, resulting in a significant reduction in yield components. On the contrary, for Syndiouk, D117, Td7 and Utique genotypes, a solute accumulation and an increase in cell wall rigidity were the main mechanisms developed during drought stress. These mechanisms were efficient in enhancing soil water uptake, limiting leaf water loss and protecting cells membranes against leakage induced by oxidative damages. Furthermore, leaf soluble sugars accumulation was the major component of osmotic adjustment in drought-stressed wheat plants. The transcriptional analysis of genes involved in the final step of the ABA biosynthesis (AAO) and in the synthesis of an aquaporin (PIP2:1) revealed distinct responses to drought stress among the selected genotypes. In the resistant genotypes, PIP2:1 was significantly upregulated whereas in the sensitive ones, its expression showed only a slight induction. Conversely, the sensitive genotypes exhibited higher levels of AAO gene expression compared to the resistant genotypes. Our results suggest that drought tolerance in wheat is regulated by the interaction between the dynamics of leaf water status and stomatal behavior. Based on our findings, Syndiouk, D117, Utique and Td7, could be used in breeding programs for developing high-yielding and drought-tolerant wheat varieties.

SNP Diversity and Genetic Structure of "Rogosija", an Old Western Balkan Durum Wheat Collection.

Durum wheat landraces represent a genetic resource for the identification and isolation of new valuable genes and alleles, useful to increase the crop adaptability to climate change. Several durum wheat landraces, all denominated "Rogosija", were extensively cultivated in the Western Balkan Peninsula until the first half of the 20th century. Within the conservation program of the Montenegro Plant Gene Bank, these landraces were collected, but without being characterized. The main goal of this study was to estimate the genetic diversity of the "Rogosija collection" consisting of 89 durum accessions, using 17 morphological descriptors and the 25K Illumina single nucleotide polymorphism (SNP) array. The genetic structure analysis of the Rogosija collection showed two distinguished clusters localized in two different Montenegro eco-geographic micro-areas, characterized by continental Mediterranean climate and maritime Mediterranean climate. Data suggest that these clusters could be composed of two different Balkan durum landrace collections evolved in two different eco-geographic micro-areas. Moreover, the origin of Balkan durum landraces is discussed.

"Ectomosphere": Insects and Microorganism Interactions.

This study focuses on interacting with insects and their ectosymbiont (lato sensu) microorganisms for environmentally safe plant production and protection. Some cases help compare ectosymbiont microorganisms that are insect-borne, -driven, or -spread relevant to endosymbionts' behaviour. Ectosymbiotic bacteria can interact with insects by allowing them to improve the value of their pabula. In addition, some bacteria are essential for creating ecological niches that can host the development of pests. Insect-borne plant pathogens include bacteria, viruses, and fungi. These pathogens interact with their vectors to enhance reciprocal fitness. Knowing vector-phoront interaction could considerably increase chances for outbreak management, notably when sustained by quarantine vector ectosymbiont pathogens, such as the actual Xylella fastidiosa Mediterranean invasion episode. Insect pathogenic viruses have a close evolutionary relationship with their hosts, also being highly specific and obligate parasites. Sixteen virus families have been reported to infect insects and may be involved in the biological control of specific pests, including some economic weevils. Insects and fungi are among the most widespread organisms in nature and interact with each other, establishing symbiotic relationships ranging from mutualism to antagonism. The associations can influence the extent to which interacting organisms can exert their effects on plants and the proper management practices. Sustainable pest management also relies on entomopathogenic fungi; research on these species starts from their isolation from insect carcasses, followed by identification using conventional light or electron microscopy techniques. Thanks to the development of omics sciences, it is possible to identify entomopathogenic fungi with evolutionary histories that are less-shared with the target insect and can be proposed as pest antagonists. Many interesting omics can help detect the presence of entomopathogens in different natural matrices, such as soil or plants. The same techniques will help localize ectosymbionts, localization of recesses, or specialized morphological adaptation, greatly supporting the robust interpretation of the symbiont role. The manipulation and modulation of ectosymbionts could be a more promising way to counteract pests and borne pathogens, mitigating the impact of formulates and reducing food insecurity due to the lesser impact of direct damage and diseases. The promise has a preventive intent for more manageable and broader implications for pests, comparing what we can obtain using simpler, less-specific techniques and a less comprehensive approach to Integrated Pest Management (IPM).

Sustainable strategies for management of the "false root-knot nematode" Nacobbus spp.

The genus Nacobbus, known as the false root-knot nematode, is native to the American continent and comprises polyphagous species adapted to a wide range of climatic conditions. Alone or in combination with other biotic and abiotic factors, Nacobbus spp. can cause significant economic yield losses on main food crops such as potato, sugar beet, tomato, pepper and bean, in South and North America. Although the genus distribution is restricted to the American continent, it has quarantine importance and is subject to international legislation to prevent its spread to other regions, such as the European Union. The management of Nacobbus spp. remains unsatisfactory due to the lack of information related to different aspects of its life cycle, survival stages in the soil and in plant material, a rapid and reliable diagnostic method for its detection and the insufficient source of resistant plant genotypes. Due to the high toxicity of chemical nematicides, the search for alternatives has been intensified. Therefore, this review reports findings on the application of environmentally benign treatments to manage Nacobbus spp. Biological control strategies, such as the use of different organisms (mainly bacteria, fungi and entomopathogenic nematodes) and other eco-compatible approaches (such as metabolites, essential oils, plant extracts, phytohormones and amendments), either alone or as part of a combined control strategy, are discussed. Knowledge of potential sources of resistance for genetic improvement for crops susceptible to Nacobbus spp. are also reported. The sustainable strategies outlined here offer immediate benefits, not only to counter the pathogen, but also as good alternatives to improve crop health and growth.

Exploring Anastomosis of Hyphae and Mating-Type Compatibility of Pochonia chlamydosporia Isolates of the Meloidogyne, Heterodera and Globodera Biotypes.

The endophytic and nematophagous fungus Pochonia chlamydosporia is an efficient biological control agent of plant-parasitic nematodes. Isolates of the fungus can be allocated to a biotype group according to the nematode host, but it is unknown if genetic interchange can occur between different biotypes, which may affect their parasitic performance. An anastomosis assay was conducted in vitro to assess hyphae vegetative compatibility/incompatibility followed by a PCR-based mating-type assay genotyping of five isolates of P. chlamydosporia var. chlamydoporia of the Meloidogyne sp. (Pc10, Pc190, Pc309), Globodera sp. (Pc280) and Heterodera avenae (Pc60) biotypes, including 16 pairwise isolates combinations in four replicates. Pairwise combinations were tested on glass slides and mycelia were stained to confirm nuclei migration between anastomosing hyphae using fluorescence microscopy. Anastomosis only occurred between mycelium hyphae of the same isolate and biotype. Mating-type PCR-based molecular assays showed that all isolates were heterothallic. The MAT1-1 genotype was found in isolates Pc10, Pc190, Pc280, Pc309, and the MAT1-2 genotype in Pc60. The results showed a vegetative incompatibility among isolates, suggesting the occurrence of such interactions for their respective biotypes. Anastomosis and PCR mating-type results suggest that different fungal biotypes can occur in the same niche but that genetic incompatibility mechanisms, such as mating-type, may limit or impede viable heterokaryosis.

Serendipitous In Situ Conservation of Faba Bean Landraces in Tunisia: A Case Study.

Cultivation of faba bean (Vicia faba L.) in Tunisia is largely based on improved varieties of the crop. However, a few farmers continue to produce local cultivars or landraces. The National Gene Bank of Tunisia (NGBT) recently launched a collection project for faba bean landraces, with special focus on the regions of the North West, traditionally devoted to cultivating grain legumes, and where around 80% of the total national faba bean cultivation area is located. The seed phenotypic features of the collected samples were studied, and the genetic diversity and population structure analyzed using simple sequence repeat markers. The genetic constitution of the present samples was compared to that of faba bean samples collected by teams of the International Center for Agricultural Research in the Dry Areas (ICARDA) in the 1970s in the same region, and stored at the ICARDA gene bank. The results of the diversity analysis demonstrate that the recently collected samples and those stored at ICARDA largely overlap, thus demonstrating that over the past 50 years, little genetic change has occurred to the local faba bean populations examined. These findings suggest that farmers serendipitously applied international best practices for in situ conservation of agricultural crops.

Potato Virus Y Infection Alters Small RNA Metabolism and Immune Response in Tomato.

Potato virus Y (PVY) isolate PVYC-to induces growth reduction and foliar symptoms in tomato, but new vegetation displays symptom recovery at a later stage. In order to investigate the role of micro(mi)RNA and secondary small(s)RNA-regulated mechanisms in tomato defenses against PVY, we performed sRNA sequencing from healthy and PVYC-to infected tomato plants at 21 and 30 days post-inoculation (dpi). A total of 792 miRNA sequences were obtained, among which were 123 canonical miRNA sequences, many isomiR variants, and 30 novel miRNAs. MiRNAs were mostly overexpressed in infected vs. healthy plants, whereas only a few miRNAs were underexpressed. Increased accumulation of isomiRs was correlated with viral infection. Among miRNA targets, enriched functional categories included resistance (R) gene families, transcription and hormone factors, and RNA silencing genes. Several 22-nt miRNAs were shown to target R genes and trigger the production of 21-nt phased sRNAs (phasiRNAs). Next, 500 phasiRNA-generating loci were identified, and were shown to be mostly active in PVY-infected tissues and at 21 dpi. These data demonstrate that sRNA-regulated host responses, encompassing miRNA alteration, diversification within miRNA families, and phasiRNA accumulation, regulate R and disease-responsive genes. The dynamic regulation of miRNAs and secondary sRNAs over time suggests a functional role of sRNA-mediated defenses in the recovery phenotype.

Characterization and potential of plant growth promoting rhizobacteria isolated from native Andean crops.

Bacteria isolated from soil and rhizosphere samples collected in Peru from Andean crops were tested in vitro and in vivo to determine their potential as plant growth promoters and their ability to induce systemic resistance to Alternaria alternata in tomato plants. The isolates were identified by sequencing their 16S ribosomal RNA gene. Test for phosphate solubilization, and indolacetic acid were also carried out, together with in vitro antagonism assays in dual cultures towards the plant pathogens Fusarium solani, A. alternata and Curvularia lunata. The three most promising isolates (Pa15, Ps155, Ps168) belonged to the genus Pseudomonas. Further assays were carried out with tomato plants to assess their plant protection effect towards A. alternata and as growth promoters. Inoculation of tomato seeds with all isolates significantly enhanced seed germination, plantlets emergence and plant development. Bacterial inoculation also reduce damage level caused by A. alternata. The expression levels of three tomato genes involved in the jasmonate (AOS), ethylene responsive (ERF-2) and pathogenesis related (PR-P2) pathways were determined in plants challenged with A. alternata, alone or with each bacterial isolate, respectively. Results showed that at 24 h after infection, in absence of the pathogen, the expression level of the tested genes was very low. The presence of A. alternata alone and in combination with bacteria increased the transcripts of all genes. Data showed a potential of best performing isolate Ps168 to sustain tomato plants nutrition and activate defense-related genes for protection by pathogenic fungi.

Pochonia chlamydosporia: Advances and Challenges to Improve Its Performance as a Biological Control Agent of Sedentary Endo-parasitic Nematodes.

The nematophagous fungus Pochonia chlamydosporia var. chlamydosporia is one of the most studied biological control agents against plant (semi-) endo-parasitic nematodes of the genera Globodera, Heterodera, Meloidogyne, Nacobbus and, more recently, Rotylenchulus. In this paper we present highlights from more than three decades of worldwide research on this biological control agent. We cover different aspects and key components of the complex plant-fungus-nematode tri-trophic interaction, an interaction that needs to be addressed to ensure the efficient use of P. chlamydosporia as a biopesticide as part of an integrated pest management approach.

In-silico and in-vivo analyses of EST databases unveil conserved miRNAs from Carthamus tinctorius and Cynara cardunculus.

BACKGROUND: MicroRNAs (miRNAs) are small RNAs (21-24 bp) providing an RNA-based system of gene regulation highly conserved in plants and animals. In plants, miRNAs control mRNA degradation or restrain translation, affecting development and responses to stresses. Plant miRNAs show imperfect but extensive complementarity to mRNA targets, making their computational prediction possible, useful when data mining is applied on different species. In this study we used a comparative approach to identify both miRNAs and their targets, in artichoke and safflower. RESULTS: Two complete expressed sequence tags (ESTs) datasets from artichoke (3.6 · 10(4) entries) and safflower (4.2 · 10(4)), were analysed with a bioinformatic pipeline and in vitro experiments, identifying 17 potential miRNAs. For each EST, using RNAhybrid program and 953 non redundant miRNA mature sequences, available in mirBase as reference, we searched matching putative targets. 8730 out of 42011 ESTs from safflower and 7145 of 36323 ESTs from artichoke showed at least one predicted miRNA target. BLAST analysis showed that 75% of all ESTs shared at least a common homologous region (E-value < 10(-4)) and about 50% of these displayed 400 bp or longer aligned sequences as conserved homologous/orthologous (COS) regions. 960 and 890 ESTs of safflower and artichoke organized in COS shared 79 different miRNA targets, considered functionally conserved, and statistically significant when compared with random sequences (signal to noise ratio > 2 and specificity ≥ 0.85). Four highly significant miRNAs selected from in silico data were experimentally validated in globe artichoke leaves. CONCLUSIONS: Mature miRNAs and targets were predicted within EST sequences of safflower and artichoke. Most of the miRNA targets appeared highly/moderately conserved, highlighting an important and conserved function. In this study we introduce a stringent parameter for the comparative sequence analysis, represented by the identification of the same target in the COS region. After statistical analysis 79 targets, found on the COS regions and belonging to 60 miRNA families, have a signal to noise ratio > 2, with ≥ 0.85 specificity. The putative miRNAs identified belong to 55 dicotyledon plants and to 24 families only in monocotyledon.

Transcriptome analysis shows differential gene expression in the saprotrophic to parasitic transition of Pochonia chlamydosporia.

Expression profiles were identified in the fungus Pochonia chlamydosporia, a biological control agent of plant parasitic nematodes, through a cDNA-amplified fragment length polymorphism approach. Two isolates with different host ranges, IMI 380407 and IMI 331547, were assayed in conditions of saprotrophic-to-parasitic transition, through in vitro assays. Gene expression profiles from three different nutritional conditions and four sampling times were generated, with eggs of host nematodes Globodera pallida and Meloidogyne incognita. Expression of transcripts changed in RNA fingerprints obtained under different nutritional stresses (starvation in presence/absence of eggs, or rich growth media). Transcript derived fragments (TDFs) obtained from the expression profiles corresponded to 6,800 products. A subset was sequenced and their expression profile confirmed through RT PCR. A total of 57 TDFs were selected for further analysis, based on similarities to translated or annotated sequences. Genes expressed during egg parasitism for both IMI 380407 and IMI 331547 were involved in metabolic functions, cellular signal regulation, cellular transport, regulation of gene expression, DNA repair, and other unknown functions. Multivariate analysis of TDF expression showed three groups for IMI 380407 and one for IMI 331547, each characterized by expression of genes related to eggs parasitism. Common amplification profiles among TDF clusters from both isolates also reflected a pool of constitutive genes, not affected by the nutritional conditions and nematode associations, related to general metabolic functions. The differential expression of parasitism related genes suggest a network of induced/repressed products, playing a role in fungal signaling and infection, with partial overlaps in host infection and parasitism traits.

Characterization of the interactions between Cucumber mosaic virus and Potato virus Y in mixed infections in tomato.

Mixed infection with the SON41 strain of Potato virus Y (PVY-SON41) in tomato increased accumulation of RNAs of strains Fny and LS of Cucumber mosaic virus (CMV-Fny and CMV-LS, respectively) and enhanced disease symptoms. By contrast, replication of PVY-SON41 was downregulated by CMV-Fny and this was due to the CMV-Fny 2b protein. The CMV-FnyΔ2b mutant was unable to systemically invade the tomato plant because its movement was blocked at the bundle sheath of the phloem. The function needed for invading the phloem was complemented by PVY-SON41 in plants grown at 22°C whereas this complementation was not necessary in plants grown at 15°C. Mutations in the 2b protein coding sequence of CMV-Fny as well as inhibition of translation of the 2a/2b overlapping region of the 2a protein lessened both the accumulation of viral RNAs and the severity of symptoms. Both of these functions were complemented by PVY-SON41. Infection of CMV-Fny supporting replication of the Tfn-satellite RNA reduced the accumulation of CMV RNA and suppressed symptom expression also in plants mixed-infected with PVY-SON41. The interaction between CMV and PVY-SON41 in tomato exhibited different features from that documented in other hosts. The results of this work are relevant from an ecological and epidemiological perspective due to the frequency of natural mixed infection of CMV and PVY in tomato.

Analysis of mechanisms involved in the Cucumber mosaic virus satellite RNA-mediated transgenic resistance in tomato plants.

Transgenic tomato (Lycopersicon esculentum Mill. cv. UC82) plants expressing a benign variant of Cucumber mosaic virus satellite RNA (CMV Tfn-satRNA) were generated. The transformed plants did not produce symptoms when challenged with a satRNA-free strain of CMV (CMV-FL). The same plant lines initially were susceptible to necrosis elicited by a CMV strain supporting a necrogenic variant of satRNA (CMV-77), but a phenotype of total recovery from the necrosis was observed in the newly developing leaves. The features of the observed resistance were analyzed and are consistent with two different mechanisms of resistance. In transgenic plants inoculated with CMV-FL strain, the symptomless phenotype was correlated to the down-regulation of CMV by Tfn-satRNA, amplified from the transgene transcripts, as the first resistance mechanism. On the other hand, the delayed resistance to CMV-77 in transgenic tomato lines was mediated by a degradation process that targets satRNAs in a sequence-specific manner. Evidence is provided for a correlation between a reduced accumulation level of transgenic messenger Tfn-satRNA, the accumulation of small (approximately 23 nucleotides) RNAs with sequence homology to satRNAs, the progressively reduced accumulation of 77-satRNA in infected tissues, and the transition in infected plants from diseased to healthy. Thus, events leading to the degradation of satRNA sequences indicate a role for RNA silencing as the second mechanism determining resistance of transgenic tomato lines.