Differences in interactions of aboveground and belowground herbivores on the invasive plant <Emphasis Type="Italic">Alternanthera philoxeroides</Emphasis> and native host <Emphasis Type="Italic">A. sessilis</Emphasis>

Plant invasions may result in novel plant-herbivore interactions. However, we know little about whether and how invasive plants can mediate native above- and belowground herbivore interactions. In this study, we conducted greenhouse experiments to examine the interaction between a native defoliating beetle, Cassida piperata, and a native root-knot nematode, Meloidogyne incognita, on the invasive alligator weed, Alternanthera philoxeroides. We also included their native host A. sessilis in the experiments to examine whether the patterns of above- and belowground herbivore interaction vary with host plants (invasive vs. native). We analyzed total carbon and nitrogen in leaves and roots attacked by M. incognita and C. piperata. M. incognita slightly negatively affected feeding by C. piperata on A. philoxeroides, and the leaf area damaged decreased as the number of M. incognita increased. M. incognita had a negative impact on total leaf nitrogen, but had no impact on total leaf carbon. M. incognita egg production on A. philoxeroides roots decreased as the amount of damage caused by C. piperata increased. Herbivory by C. piperata did not affect total root carbon or nitrogen. M. incognita and C. piperata did not affect each other on the native plant A. sessilis. These results suggest that invasive plants can mediate native above- and belowground herbivore interactions. The knowledge of how invasive plants affect those interactions is crucial for better understanding the impacts of biological invasions on native above- and belowground organisms.     

Expression of plant antimicrobial peptide <Emphasis Type="Italic">pro-SmAMP2</Emphasis> gene increases resistance of transgenic potato plants to <Emphasis Type="Italic">Alternaria</Emphasis> and <Emphasis Type="Italic">Fusarium</Emphasis> pathogens

The chickweed (Stellaria media L.) pro-SmAMP2 gene encodes the hevein-like peptides that have in vitro antimicrobial activity against certain harmful microorganisms. These peptides play an important role in protecting the chickweed plants from infection, and the pro-SmAMP2 gene was previously used to protect transgenic tobacco and Arabidopsis plants from phytopathogens. In this study, the pro-SmAMP2 gene under control of viral CaMV35S promoter or under control of its own pro-SmAMP2 promoter was transformed into cultivated potato plants of two cultivars, differing in the resistance to Alternaria: Yubiley Zhukova (resistant) and Skoroplodny (susceptible). With the help of quantitative real-time PCR, it was demonstrated that transgenic potato plants expressed the pro-SmAMP2 gene under control of both promoters at the level comparable to or exceeding the level of the potato actin gene. Assessment of the immune status of the transformants demonstrated that expression of antimicrobial peptide pro-SmAMP2 gene was able to increase the resistance to a complex of Alternaria sp. and Fusarium sp. phytopathogens only in potato plants of the Yubiley Zhukova cultivar. The possible role of the pro-SmAMP2 products in protecting potatoes from Alternaria sp. and Fusarium sp. is discussed.     

Stimulation of Cellular Mechanisms of Potato Antivirus Resistance by the Action of a Preparation Based on <Emphasis Type="Italic">Bacillus subtilis</Emphasis> Bacteria

A comparative study of the antiviral activity of a new biopreparation based on bacteria Bacillus subtilis (strain 47) and commercial biopesticides (beta-protectin, phyto-protectin, frutin) in potato plants of Belarusian selection (Lileya and Scarb) was carried out in vivo and ex vitro. Pretreatment of plants with B. subtilis biopreparation and biopesticides prevents infection by potato X-viruses. The antiviral efficacy of B. subtilis does not depend on the conditions of plant growth and is more effective than biopesticides. Increased potato plant resistance to viral infection was accompanied by an effect leading to an increase in the mass of minitubers and the dry matter content in them. Treatment of potato plants with B. subtilis did not affect the molecular heterogeneity of peroxidase and superoxide dismutase, but it changed the relative activity of their isoforms. Treatment with a bacterial preparation increased the activity of superoxide dismutase when it was applied both to intact plants and those preinfected with virus. The results indicate that pretreatment of potato plants with the B. subtilis drug prevents virus infection, inducing the antiviral resistance of the potato, and is accompanied by a change in the activity of redox enzymes.     

Physiological and biochemical changes in tomato cultivar PT-3 with dual inoculation of mycorrhiza and PGPR against root-knot nematode

Arbuscular mycorrhizal fungi (AMF) and plant growth promoting rhizobacteria (PGPR) have potential to control soil-borne diseases including plant-parasitic nematodes. First, the effects of dual inoculation of mycorrhiza (Rhizophagus irregularis) and two stains of pseudomonads (Pseudomonas jessenii strain R62 and Pseudomonas synxantha strain R81) on tomato (Solanum lycopersicum cv. PT-3) growth were tested. Further, the physiological and biochemical changes caused by these beneficial organisms during infection by the root-knot nematode Meloidogyne incognita were studied. The experiment was conducted under glass house conditions and carried out up to one month after nematode inoculation. Plants treated with dual or individual inoculation of AMF and PGPR showed significantly enhanced plant growth and reduced nematode infection. In addition, they exhibited potent activity of phenolics (28 %) and defensive enzymes i.e. peroxidase (PO; 1.26 fold), polyphenyloxidase (PPO; 1.35 fold) and superoxide dismutase (SOD; 1.09 fold) while a significant reduction in malondialdehyde (MDA; 1.63 fold) and hydrogen peroxide (H₂O₂; 1.30 fold) content was recorded when compared to the nematode-infected plants. These findings indicate the feasibility of AMF and PGPR individually or in combinations as potential biocontrol agents for the management of root-knot nematodes.     

Root nematode infection enhances leaf defense against whitefly in tomato

The foliar response to different herbivores sharing the same hosts is an important topic for the study of plant-insect interactions. Plants evolve local and systemic resistant strategies to cope with herbivores. Many researchers have characterized the mechanisms of leaf responses to insect infestation; however, the fact that roots serve as systemic resistance modulators to leaf herbivores has been widely ignored. Here, we report that tomato (Solanum lycopersicum) plants infected with southern root-knot nematodes (Meloidogyne incognita)—which feed on the roots to form nodules—enhanced leaf defenses against aboveground attackers, specifically, the whitefly (Bemisia tabaci). Our results show that nematode infection reduced the whitefly population abundance because of conferring a stronger SA-dependent defense pathway against whitefly than in tomato plants without nematode infection. Meanwhile, nematode-infected tomato plant also activated the foliar JA-dependent defense pathway at 4 h after whitefly infestation. However, the foliar JA-dependent defense under whitefly infestation alone was suppressed, with the JA content being nearly 30 % lower than that in tomato plants co-infected with nematodes and whiteflies. Furthermore, nematode infection significantly decreased the plant nitrogen concentration in leaves and roots. As a result, nematode infection reduced the number of whiteflies by enhancing foliar SA-dependent defense, activating JA-dependent defense and decreasing nitrogen nutrition. Our results suggest that underground nematode infection significantly enhances the defense ability of tomato plants against whitefly.     

Genetic analyses of resistance to the peach root-knot nematode (<Emphasis Type="Italic">Meloidogyne floridensis</Emphasis>) using microsatellite markers

Most commercially important rootstocks for peach [Prunus persica (L.) Batsch] had been selected for resistance to one or more of the root-knot nematode (RKN) species: Meloidogyne incognita, M. arenaria, and M. javanica. The peach root-knot nematode, M. floridensis (MF), is a relatively newly discovered threat to peach and is not controlled by resistance genes in “Nemared,” “Nemaguard,” and “Okinawa.” The “Flordaguard” peach seedling rootstock, conventionally bred to provide resistance to MF, has solely been used for low-chill peach production in Florida for over 20 years and has already shown signs of resistance breakdown. A source of high resistance to the pathogenic MF isolate (“MFGnv14”) was identified from wild peach Prunus kansuensis Rehder (Kansu peach), thereby suggesting the potential for broadening spectrum and increasing durability of resistance in peach rootstocks through interspecific hybridization with P. kansuensis. Using 12 F₂ and BC₁F₁ populations derived from crosses between Okinawa or Flordaguard peach and P. kansuensis populations, we examined the genetic control for MF resistance by identifying associated microsatellite markers and determining genomic location of the resistance locus. One microsatellite marker (UDP98-025) showed strong and consistent association with resistance based on root-galling index. The resistance locus was mapped on the subtelomeric region of linkage group 2, co-localizing with other previously reported RKN resistance genes in Prunus. Segregation of gall-index-based resistance observed in F₂ and BC₁F₁ populations is compatible with the involvement of a multiallelic locus wherein a dominant (Mf₁) or recessive (mf₃) resistance allele is inherited from P. kansuensis, and susceptibility alleles (mf₂) from peach.     

Overexpression of barley nicotianamine synthase 1 confers tolerance in the sweet potato to iron deficiency in calcareous soil

Background and aims

Iron (Fe) is an essential micronutrient for all higher organisms. Fe is sparingly available in calcareous soils and Fe deficiency is a major agricultural problem worldwide. Nicotianamine (NA) is a metal chelator involved in metal translocation in plants. Sweet potato is an attractive crop that can grow in poor soil and thus is useful for planting in uncultivated soil. In addition, the sweet potato has recently been suggested as a source of bioethanol. Our aim is to increase NA concentration in sweet potato to ameliorate Fe deficiency.

Method

Sweet potato plants expressing the barley NA synthase 1 (HvNAS1) gene under the control of CaMV 35S promoter were produced by Agrobacterium-mediated transformation.

Results

The transgenic sweet potato exhibited tolerance to low Fe availability when grown in calcareous soil. The level of tolerance to low Fe availability was positively correlated with the HvNAS1 expression level. The NA concentration of the transgenic sweet potato leaves was up to 7.9-fold greater than that of the non-transgenic (NT) plant leaves. Furthermore, the Fe and zinc concentrations were 3- and 2.9-fold greater, respectively, in transgenic sweet potato than in NT plant leaves.

Conclusions

Our results suggest that increasing the NA concentration of sweet potato by overexpression of HvNAS1 could significantly improve agricultural productivity and energy source.

     

Identification and characterization of microRNAs in the plant parasitic root-knot nematode <Emphasis Type="Italic">Meloidogyne incognita</Emphasis> using deep sequencing

The root-knot nematode Meloidogyne incognita is among the most damaging plant-parasitic pests of several crops including cotton (Gossypium hirsutum) and tomato (Lycopersicon escultentum). Recently, a genome has become available for M. incognita, which greatly facilitates investigation of the interactions between M. incognita and its plant hosts at the molecular level and enables formation of hypotheses concerning development at the cellular level. MicroRNAs (miRNAs) are a class of small RNA molecules that serve as endogenous gene regulators. They regulate many biological processes including reproduction, the sequencing of morphological development, and potentially of parasitism as well. Certain miRNAs regulate fundamental metabolism pathways and stress responses in M. incognita. Since a list of miRNAs has not been generated for M. incognita, we employed a bioinformatics tool called mirDeepFinder to identify miRNAs from the small RNA database of M. incognita (GSM611102) that was generated from deep sequencing. A total of 254 conserved miRNAs belonging to 161 miRNA families were identified, as were 35 novel miRNAs belonging to 31 families. The 16 most commonly found miRNAs in order of abundance were min-miR-100a, min-miR-124, min-miR-71a, min-miR-1, min-miR-228, min-miR-92, min-miR-72, min-miR-49b, min-miR-58, min-miR-252, min-miR-lin-4, min-miR-87, min-miR-2a, min-miR-34a, min-miR-50a, and min-miR-279a. The length of the pre-miRNAs varied greatly from 50 to 197 nt, with an average of 88?±?39 nt. The average minimal folding free energy (MFE) and MFE index (MFEI) of the identified miRNAs were –30.3 Kcal/mol and 0.92, respectively, indicating that these miRNAs can readily fold into a typical hairpin secondary structure.     

Effector-activated immune responses in potato and tobacco cell cultures caused by phytopathogen <Emphasis Type="Italic">Clavibacter michiganensis</Emphasis> ssp. <Emphasis Type="Italic">sepedonicus</Emphasis>

Generation of reactive oxygen species (ROS) in tobacco (Nicotiana tabacum L.) cell cultures and potato (Solanum tuberosum L.) of two varieties experiencing the action of bacterial pathogen Clavibacter michiganensis ssp. sepedonicus (Cms) was investigated. The intensity and dynamics of the changes in hydrogen peroxide concentration observed in these cultures provided evidence for the development in tobacco of the effector-activated immune responses and the induction of the same type of responses but with low intensity for resistant potato variety and the inhibition of the defense mechanisms for its susceptible variety. This is in accordance with the data concerning the dynamics of plant cell culture death as well as the results obtained earlier on the whole plants. The experiments performed had also the purpose to elucidate whether the development of the above responses on the ability of bacteria Cms to form biofilms during plant infection. It was shown that this ability of Cms is significantly inhibited upon the combined cultivation of it with the plant cells exerting the responses of the effector-activated immunity and represented by the cells of tobacco and resistant potato variety. In the case of susceptible potato variety, the process of the biofilm formation was suppressed by the plant only to a slight extent. In addition, the fact concerning the participation of heat shock proteins (HSPs) in the development of the effector-activated immune responses was revealed.     

Down-regulation of <Emphasis Type="Italic">Arabidopsis</Emphasis><Emphasis Type="Italic">DND1</Emphasis> orthologs in potato and tomato leads to broad-spectrum resistance to late blight and powdery mildew

Multiple susceptibility genes (S), identified in Arabidopsis, have been shown to be functionally conserved in crop plants. Mutations in these S genes result in resistance to different pathogens, opening a new way to achieve plant disease resistance. The aim of this study was to investigate the role of Defense No Death 1 (DND1) in susceptibility of tomato and potato to late blight (Phytophthora infestans). In Arabidopsis, the dnd1 mutant has broad-spectrum resistance against several fungal, bacterial, and viral pathogens. However this mutation is also associated with a dwarfed phenotype. Using an RNAi approach, we silenced AtDND1 orthologs in potato and tomato. Our results showed that silencing of the DND1 ortholog in both crops resulted in resistance to the pathogenic oomycete P. infestans and to two powdery mildew species, Oidium neolycopersici and Golovinomyces orontii. The resistance to P. infestans in potato was effective to four different isolates although the level of resistance (complete or partial) was dependent on the aggressiveness of the isolate. In tomato, DND1-silenced plants showed a severe dwarf phenotype and autonecrosis, whereas DND1-silenced potato plants were not dwarfed and showed a less pronounced autonecrosis. Our results indicate that S gene function of DND1 is conserved in tomato and potato. We discuss the possibilities of using RNAi silencing or loss-of-function mutations of DND1 orthologs, as well as additional S gene orthologs from Arabidopsis, to breed for resistance to pathogens in crop plants.     

Cloning and characterization of the new multiple stress responsible gene I (<Emphasis Type="Italic">MuSI</Emphasis>) from sweet potato

New multiple-stress related gene isolated from sweet potato and designated it as MusI (multiple stress responsible gene I). Sequence analysis revealed that its full length cDNA was 998 bp long and included a 717 bp open reading frame encoding for 238 amino acids. Comparison of its cDNA and genomic DNA sequence showed that 3 exons were divided by 2 introns in its ORF region. Its deduced amino acid sequence contained a conserved rubber elongation factor (REF) domain and showed high homology with many stress-related proteins. Therefore, it was named MuSI (multiple stress responsible gene I). Southern hybridization analysis indicated that the MuSI gene may belong to a multi-gene family. Expression pattern of the MuSI gene showed that it was differently expressed among roots, stems, leaves, and flowers of a sweet potato, and its expression level was especially high in flowers and white fibrous roots. Its expression was also highly induced by various stress signals including dehydration, high salt, heavy metal, oxidation, and plant hormones. Stress tolerance experiment using transgenic plants overexpressing the MuSI gene showed that all independent transgenic tobacco lines have enhanced tolerance to high temperature stress. Among them, transgenic line 6 particularly showed tolerance to salt, heavy metal, and osmotic stress as well. These results suggest that the MuSI gene functions as a positive regulator of various stress responses and may be useful in improving stress tolerance of transgenic plants.     

Detection of symbionts and virus in the whitefly <Emphasis Type="Italic">Bemisia tabaci</Emphasis> (Hemiptera: Aleyrodidae), vector of the <Emphasis Type="Italic">Mungbean yellow mosaic India virus</Emphasis> in Central India

Legume crops in Central India, the main soybean production area of the country, may suffer from yellow mosaic disease caused by the Mungbean yellow mosaic India virus (MYMIV). MYMIV is transmitted by the sweet potato whitefly, Bemisia tabaci (Gennadius), which is a species complex composed of various genetic groups. This vector species harbors different endosymbionts among regional strains and among individuals. To elucidate fundamental aspects of this virus vector in the state of Madhya Pradesh, the infection status of the symbionts and the virus in whiteflies was studied. A polymerase chain reaction (PCR) survey of the whiteflies collected in Madhya Pradesh found four secondary endosymbionts, Arsenophonus, Hemipteriphilus, Wolbachia, and Cardinium, in addition to the primary endosymbiont Portiera. Arsenophonus and Hemipteriphilus were highly infected but the infection rates of Wolbachia and Cardinium were low. MYMIV was detected in whitefly populations collected from various host plants in Madhya Pradesh. The whitefly populations belonged to the Asia I and II genetic groups; several different Asia II populations were also distributed. Specific relations were not observed among symbiont infection status, virus infection, and the whitefly genetic groups in the populations of Madhya Pradesh, though Cardinium was highly detected in the Asia II-1 group. New primers, which can be used for PCR template validation and for discriminating two phylogenetically close endosymbionts, were designed.     

Fungal root endophytes of tomato from Kenya and their nematode biocontrol potential

The significance of fungal endophytes in African agriculture, particularly Kenya, has not been well investigated. Therefore, the objective of the present work was isolation, multi-gene phylogenetic characterization and biocontrol assessment of endophytic fungi harbored in tomato roots for nematode infection management. A survey was conducted in five different counties along the central and coastal regions of Kenya to determine the culturable endophytic mycobiota. A total of 76 fungal isolates were obtained and characterized into 40 operational taxonomic units based on the analysis of ITS, β-tubulin and tef1α gene sequence data. Among the fungal isolates recovered, the most prevalent species associated with tomato roots were members of the Fusarium oxysporum and F. solani species complexes. Of the three genes utilized for endophyte characterization, tef1α provided the best resolution. A combination of ITS, β-tubulin and tef1α resulted in a better resolution as compared to single gene analysis. Biotests demonstrated the ability of selected non-pathogenic fungal isolates to successfully reduce nematode penetration and subsequent galling as well as reproduction of the root-knot nematode Meloidogyne incognita. Most Trichoderma asperellum and F. oxysporum species complex isolates reduced root-knot nematode egg densities by 35–46 % as compared to the non-fungal control and other isolates. This study provides first insights into the culturable endophytic mycobiota of tomato roots in Kenya and the potential of some isolates for use against the root-knot nematode M. incognita. The data can serve as a framework for fingerprinting potential beneficial endophytic fungal isolates which are optimized for abiotic and biotic environments and are useful in biocontrol strategies against nematode pests in Kenyan tomato cultivars. This information would therefore provide an alternative or complementary crop protection component.     

Natural variation of potato <Emphasis Type="Italic">allene oxide synthase 2</Emphasis> causes differential levels of jasmonates and pathogen resistance in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Natural variation of plant pathogen resistance is often quantitative. This type of resistance can be genetically dissected in quantitative resistance loci (QRL). To unravel the molecular basis of QRL in potato (Solanum tuberosum), we employed the model plant Arabidopsis thaliana for functional analysis of natural variants of potato allene oxide synthase 2 (StAOS2). StAOS2 is a candidate gene for QRL on potato chromosome XI against the oömycete Phytophthora infestans causing late blight, and the bacterium Erwinia carotovora ssp. atroseptica causing stem black leg and tuber soft rot, both devastating diseases in potato cultivation. StAOS2 encodes a cytochrome P450 enzyme that is essential for biosynthesis of the defense signaling molecule jasmonic acid. Allele non-specific dsRNAi-mediated silencing of StAOS2 in potato drastically reduced jasmonic acid production and compromised quantitative late blight resistance. Five natural StAOS2 alleles were expressed in the null Arabidopsis aos mutant under control of the Arabidopsis AOS promoter and tested for differential complementation phenotypes. The aos mutant phenotypes evaluated were lack of jasmonates, male sterility and susceptibility to Erwinia carotovora ssp. carotovora. StAOS2 alleles that were associated with increased disease resistance in potato complemented all aos mutant phenotypes better than StAOS2 alleles associated with increased susceptibility. First structure models of ‘quantitative resistant’ versus ‘quantitative susceptible’ StAOS2 alleles suggested potential mechanisms for their differential activity. Our results demonstrate how a candidate gene approach in combination with using the homologous Arabidopsis mutant as functional reporter can help to dissect the molecular basis of complex traits in non model crop plants.