Early characterization of somatic hybrids from symmetric protoplast electrofusion of <Emphasis Type="Italic">Solanum pinnatisectum</Emphasis> Dun. and <Emphasis Type="Italic">Solanum tuberosum</Emphasis> L.

Variability of 31 somatic hybrids of Solanum pinnatisectum Dun. with Solanum tuberosum L. for leaf morphology, plant vigor, resistance to Phytophthora infestans, ploidy level, and cytoplasm type was evaluated in vitro. The composition of these somatic hybrids was as follows: [S. pinnatisectum Dun. (2n = 2x = 24; cytoplasmic type Wγ) + S. tuberosum L. (2n = 4x = 48; cytoplasmic type Tß)]. Based on leaf morphology and plant growth vigor, plants were divided into three groups, including plants close to tbr parent with unlobed leaves, small plants with scarcely dissected leaves, and vigorous plants with asymmetrically and pinnately lobed leaves. Nine of the somatic hybrids were found to be highly resistant to P. infestans. Somatic hybrids were either tetraploid or hexaploid, with hexaploids being predominant. The cytoplasm of somatic hybrids was either Tßγ or Wßγ, with Tßγ being more common. Overall, in contrast to leaf morphology and growth vigor, level of resistance to P. infestans was not related to either ploidy level or type of cytoplasm. These findings demonstrate that early in vitro selection of promising hybrids can be useful in breeding programs.     

Identification of <Emphasis Type="Italic">Bacillus</Emphasis> Probiotics Isolated from Soil Rhizosphere Using 16S rRNA, <Emphasis Type="Italic">recA</Emphasis>, <Emphasis Type="Italic">rpoB</Emphasis> Gene Sequencing and RAPD-PCR

Some Bacillus species, especially Bacillus subtilis and Bacillus pumilus groups, have highly similar 16S rRNA gene sequences, which are hard to identify based on 16S rDNA sequence analysis. To conquer this drawback, rpoB, recA sequence analysis along with randomly amplified polymorphic (RAPD) fingerprinting was examined as an alternative method for differentiating Bacillus species. The 16S rRNA, rpoB and recA genes were amplified via a polymerase chain reaction using their specific primers. The resulted PCR amplicons were sequenced, and phylogenetic analysis was employed by MEGA 6 software. Identification based on 16S rRNA gene sequencing was underpinned by rpoB and recA gene sequencing as well as RAPD-PCR technique. Subsequently, concatenation and phylogenetic analysis showed that extent of diversity and similarity were better obtained by rpoB and recA primers, which are also reinforced by RAPD-PCR methods. However, in one case, these approaches failed to identify one isolate, which in combination with the phenotypical method offsets this issue. Overall, RAPD fingerprinting, rpoB and recA along with concatenated genes sequence analysis discriminated closely related Bacillus species, which highlights the significance of the multigenic method in more precisely distinguishing Bacillus strains. This research emphasizes the benefit of RAPD fingerprinting, rpoB and recA sequence analysis superior to 16S rRNA gene sequence analysis for suitable and effective identification of Bacillus species as recommended for probiotic products.     

Screening and molecular identification of lactic acid bacteria from <Emphasis Type="Italic">gari</Emphasis> and <Emphasis Type="Italic">fufu</Emphasis> and <Emphasis Type="Italic">gari</Emphasis> effluents

Bacterial strains were isolated from cassava-derived food products and, for the first time, from cassava by-products, with a focus on gari, a flour-like product, and the effluents from the production processes for gari and fufu (a dough also made from cassava flour). A total of 47 strains were isolated, all of which were tested to determine their resistance to acidic pH and to bile salt environments. Four of the 47 isolates tested positive in both environments, and these four isolates also showed antibacterial behaviour towards both Gram-positive and Gram-negative microbial pathogens (i.e. Methicillin-resistance Staphylococcus aureus, Listeria monocytogenes, Bacillus cereus, Salmonella enteritidis, Escherichia coli, Escherichia coli (O157), Yersinia enterocolitica). In most cases, the antibacterial activity was related to bacteriocin production. Molecular identification analysis (16S rDNA and randomly amplified polymorphic DNA-PCR) revealed that the four isolates were different strains of the same species, Lactobacillus fermentum. These results demonstrate that bacteria isolated from cassava-derived food items and cassava by-products have interesting properties and could potentially be used as probiotics.     

<Emphasis Type="Italic">Candida krusei</Emphasis> and <Emphasis Type="Italic">Candida glabrata</Emphasis> reduce the filamentation of <Emphasis Type="Italic">Candida albicans</Emphasis> by downregulating expression of <Emphasis Type="Italic">HWP1</Emphasis> gene

Pathogenicity of Candida albicans is associated with its capacity switch from yeast-like to hyphal growth. The hyphal form is capable to penetrate the epithelial surfaces and to damage the host tissues. Therefore, many investigations have focused on mechanisms that control the morphological transitions of C. albicans. Recently, certain studies have showed that non-albicans Candida species can reduce the capacity of C. albicans to form biofilms and to develop candidiasis in animal models. Then, the objective of this study was to evaluate the effects of Candida krusei and Candida glabrata on the morphogenesis of C. albicans. Firstly, the capacity of reference and clinical strains of C. albicans in forming hyphae was tested in vitro. After that, the expression of HWP1 (hyphal wall protein 1) gene was determined by quantitative real-time PCR (polymerase chain reaction) assay. For both reference and clinical strains, a significant inhibition of the hyphae formation was observed when C. albicans was incubated in the presence of C. krusei or C. glabrata compared to the control group composed only by C. albicans. In addition, the culture mixed of C. albicans-C. krusei or C. albicans-C. glabrata reduced significantly the expression of HWP1 gene of C. albicans in relation to single cultures of this specie. In both filamentation and gene expression assays, C. krusei showed the higher inhibitory activity on the morphogenesis of C. albicans compared to C. glabrata. C. krusei and C. glabrata are capable to reduce the filamentation of C. albicans and consequently decrease the expression of the HWP1 gene.     

Molecular insights into the origin of the brown rust resistance gene <Emphasis Type="Italic">Bru</Emphasis>1 among <Emphasis Type="Italic">Saccharum</Emphasis> species

Key message

Analysis of 387 sugarcane clones using Bru 1 diagnostic markers revealed two possible sources of Bru 1 in Chinese cultivars: one from Saccharum spontaneum and another from Saccharum robustum of New Guinea.

Abstract

Sugarcane brown rust (SBR) is an important fungal disease in many sugarcane production areas around the world, and can cause considerable yield losses in susceptible sugarcane cultivars. One major SBR resistance gene, named Bru1, initially identified from cultivar R570, was shown to be a major SBR resistance source in most of the sugarcane producing areas of the world. In this study, by using the two Bru1-associated markers, R12H16 and 9O20-F4, we surveyed the presence of Bru1 in a Chinese sugarcane germplasm collection of 387 clones, consisting of 228 hybrid cultivars bred by different Chinese sugarcane breeding establishments, 54 exotic hybrid cultivars introduced from other countries and 105 clones of sugarcane ancestral species. The Bru1-bearing haplotype was detected in 43.4% of Chinese sugarcane cultivars, 20.4% of exotic hybrid cultivars, and only 3.8% of ancestral species. Among the 33 Chinese cultivars for which phenotypes of resistance to SBR were available, Bru1 was present in 69.2% (18/26) of the resistant clones. Analyses of the allelic sequence variations of R12H16 and 9O20-F4 suggested two possible sources of Bru1 in Chinese cultivars: one from S. spontaneum and another from S. robustum of New Guinea. In addition, we developed an improved Bru1 diagnostic marker, 9O20-F4-HaeIII, which can eliminate all the false results of 9O20-F4-RsaI observed among S. spontaneum, as well as a new dominant Bru1 diagnostic marker, R12E03-2, from the BAC ShCIR12E03. Our results provide valuable information for further efforts of breeding SBR-resistant varieties, searching new SBR resistance sources and cloning of Bru1 in sugarcane.

     

A putative <Emphasis Type="Italic">NEM1</Emphasis> homologue regulates lipid droplet biogenesis <Emphasis Type="Italic">via PAH1</Emphasis> in <Emphasis Type="Italic">Tetrahymena thermophila</Emphasis>

Nuclear envelope morphology protein 1 (NEM1) along with a phosphatidate phosphatase (PAH1) regulates lipid homeostasis and membrane biogenesis in yeast and mammals. We investigated four putative NEM1 homologues (TtNEM1A, TtNEM1B, TtNEM1C and TtNEM1D) in the Tetrahymena thermophila genome. Disruption of TtNEM1B, TtNEM1C or TtNEM1D did not compromise normal cell growth. In contrast, we were unable to generate knockout strain of TtNEM1A under the same conditions, indicating that TtNEM1A is essential for Tetrahymena growth. Interestingly, loss of TtNEM1B but not TtNEM1C or TtNEM1D caused a reduction in lipid droplet number. Similar to yeast and mammals, TtNem1B of Tetrahymena exerts its function via Pah1, since we found that PAH1 overexpression rescued loss of Nem1 function. However, unlike NEM1 in other organisms, TtNEM1B does not regulate ER/nuclear morphology. Similarly, neither TtNEM1C nor TtNEM1D is required to maintain normal ER morphology. While Tetrahymena PAH1 was shown to functionally replace yeast PAH1 earlier, we observed that Tetrahymena NEM1 homologues did not functionally replace yeast NEM1. Overall, our results suggest the presence of a conserved cascade for regulation of lipid homeostasis and membrane biogenesis in Tetrahymena. Our results also suggest a Nem1-independent function of Pah1 in the regulation of ER morphology in Tetrahymena.     

<Emphasis Type="Italic">Diplazium</Emphasis> hybrids involving <Emphasis Type="Italic">D. plantaginifolium</Emphasis> and <Emphasis Type="Italic">D</Emphasis>. <Emphasis Type="Italic">ternatum</Emphasis> from Mexico and Central America

Here we evaluate the origins and relationships of Mexican and Central American Diplazium hybrids derived from crosses involving either D. plantaginifolium or D. ternatum. Based on study of live plants and herbarium specimens, we distinguish D. ×verapax from the similar D. riedelianum and present evidence that the former is a sterile hybrid derived from a cross between D. plantaginifolium and D. werckleanum. We also describe new hybrids, D. ×torresianum and D. ×subternatum from Mexico and northern Central America. Both involve D. ternatum as one parent. Diplazium. cristatum is the other putative parent of D. ×torresianum, and D. plantaginifolium is the second parent of D. ×subternatum. We also designate lectotypes for D. cordovense and D. dissimile.     

Impact of high temperature on sucrose translocation,sugar content and inulin yield in <Emphasis Type="Italic">Cichorium intybus</Emphasis> L. var. <Emphasis Type="Italic">sativum</Emphasis>

Aims

Dianthus caryophyllus is a commercially important ornamental flower. Plant growth promoting rhizobacteria are increasingly applied as bio-fertilisers and bio-fortifiers. We studied the effect of a rhizospheric isolate Klebsiella SGM 81 strain to promote D. caryophyllus growth under sterile and non-sterile conditions, to colonise its root system endophytically and its impact on the cultivatable microbial community. We identified the auxin indole-3-acetic acid (IAA) production of Klebsiella SGM 81 as major bacterial trait most likely to enhance growth of D. caryophyllus.

Methods

ipdC dependent IAA production of SGM 81 was quantified using LC-MS/MS and localised proximal to D. caryophyllus roots and correlated to root growth promotion and characteristic morphological changes. SGM 81 cells were localised on and within the plant root using 3D rendering confocal microscopy of gfp expressing SGM 81. Using Salkowski reagent IAA production was quantified and localised proximal to roots in situ. The effect of different bacterial titres on rhizosphere bacterial population was CFU enumerated on nutrient agar. The genome sequence of Klebsiella SGM 81 (accession number PRJEB21197) was determined to validate PGP traits and phylogenic relationships.

Results

Inoculation of D. caryophyllus roots with Klebsiella SGM 81 drastically promoted plant growth when grown in agar and soil, concomitant with a burst in root hair formation, suggesting an increase in root auxin activity. We sequenced the Klebsiella SGM 81 genome, identified the presence of a canonical ipdC gene in Klebsiella SGM 81, confirmed bacterial production and secretion of IAA in batch culture using LC-MS/MS and localised plant dependent IAA production by SGM 81 proximal to roots. We found Klebsiella SGM 81 to be a rhizoplane and endophytic coloniser of D. caryophyllus roots in a dose dependent manner. We found no adverse effects of SGM 81 on the overall rhizospheric microbial population unless supplied to soil in very high titres.

Conclusion

Klebsiella SGM 81 effectively improves root traits of D. caryophyllus in a dose dependent manner, likely through tryptophan dependent IAA production in the rhizoplane and potentially within the intercellular spaces of root tissue. Under optimal plant growth promoting conditions in non-sterile soil, the high total microbial titre in the rhizosphere supports a mutualistic relationship between Klebsiella SGM 81 and carnation that potentially extends to the wider rhizosphere microbiota.

     

Role of Arabidopsis <Emphasis Type="Italic">ABF1</Emphasis>/<Emphasis Type="Italic">3</Emphasis>/<Emphasis Type="Italic">4</Emphasis> during <Emphasis Type="Italic">det1</Emphasis> germination in salt and osmotic stress conditions

Key message

Arabidopsis det1 mutants exhibit salt and osmotic stress resistant germination. This phenotype requires HY5, ABF1, ABF3, and ABF4.

Abstract

While DE-ETIOLATED 1 (DET1) is well known as a negative regulator of light development, here we describe how det1 mutants also exhibit altered responses to salt and osmotic stress, specifically salt and mannitol resistant germination. LONG HYPOCOTYL 5 (HY5) positively regulates both light and abscisic acid (ABA) signalling. We found that hy5 suppressed the det1 salt and mannitol resistant germination phenotype, thus, det1 stress resistant germination requires HY5. We then queried publically available microarray datasets to identify genes downstream of HY5 that were differentially expressed in det1 mutants. Our analysis revealed that ABA regulated genes, including ABA RESPONSIVE ELEMENT BINDING FACTOR 3 (ABF3), are downregulated in det1 seedlings. We found that ABF3 is induced by salt in wildtype seeds, while homologues ABF4 and ABF1 are repressed, and all three genes are underexpressed in det1 seeds. We then investigated the role of ABF3, ABF4, and ABF1 in det1 phenotypes. Double mutant analysis showed that abf3, abf4, and abf1 all suppress the det1 salt/osmotic stress resistant germination phenotype. In addition, abf1 suppressed det1 rapid water loss and open stomata phenotypes. Thus interactions between ABF genes contribute to det1 salt/osmotic stress response phenotypes.

     

<Emphasis Type="Italic">Fusarium verticillioides</Emphasis> and fumonisin contamination in <Emphasis Type="Italic">Bt</Emphasis> and non-<Emphasis Type="Italic">Bt</Emphasis> maize cultivated in Brazil

Fusarium verticillioides is one of the main pathogens of maize, causing ear and stalk rots. This fungus is also able to produce high levels of fumonisins, which have been linked to various illnesses in humans and animals. Previous studies have shown that maize hybrids genetically modified with the cry genes from the bacterium Bacillus thuringiensis (Bt) presented lower incidence of F. verticillioides and fumonisin levels, presumably through the reduction of insects, which could act as vectors of fungi. The aim of this study was to assess the incidence of F. verticillioides and the concentration of fumonisins in Bt and isogenic non-Bt hybrids (2B710Hx, 30F35YG, 2B710, and 30F35, respectively). The samples of 2B710Hx and 30F35YG presented lower F. verticillioides frequency than 2B710 and 30F35 samples. However, there was no statistical difference between fumonisin contamination when Bt and non-Bt samples were compared (P > 0.05). The results suggest that other environmental parameters could possibly trigger fumonisin production during plant development in the field; consequently, other management strategies should be applied to aid controlling fumonisin contamination in maize.     

Strains of <Emphasis Type="Italic">Bacillus</Emphasis> ssp. regulate wheat resistance to <Emphasis Type="Italic">Septoria nodorum</Emphasis> Berk.

The ability of Bacillus subtilis Cohn and Bacillus thuringiensis Berliner to induce systemic resistance in wheat plants to the casual agent of Septoria nodorum Berk., blotch has been studied. It has been shown that strains of Bacillus ssp. that possess the capacity for endophytic survival have antagonistic activity against this pathogen in vitro. A reduction of the degree of Septoria nodorum blotch development on wheat leaves under the influence of Bacillus spp. was accompanied by the suppression of catalase activity, an increase in peroxidase activity and H₂O₂ content, and expression of defence related genes such us PR-1, PR-6, and PR-9. It has been shown that B. subtilis 26 D induces expression levels of wheat pathogenesis-related (PR) genes which marks a SA-dependent pathway of sustainable development and that B. thuringiensis V-5689 and V-6066 induces a JA/ET-dependent pathway. These results suggest that these strain Bacillus spp. promotes the formation of wheat plant resistance to S. nodorum through systemic activation of the plant defense system. The designed bacterial consortium formed a complex biological response in wheat plants infected phytopathogen.     

Selected reactive oxygen species and antioxidant enzymes in common bean after <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> pv. <Emphasis Type="Italic">phaseolicola</Emphasis> and <Emphasis Type="Italic">Botrytis cinerea</Emphasis> infection

Phaseolus vulgaris cv. Korona plants were inoculated with the bacteria Pseudomonas syringae pv. phaseolicola (Psp), necrotrophic fungus Botrytis cinerea (Bc) or with both pathogens sequentially. The aim of the experiment was to determine how plants cope with multiple infection with pathogens having different attack strategy. Possible suppression of the non-specific infection with the necrotrophic fungus Bc by earlier Psp inoculation was examined. Concentration of reactive oxygen species (ROS), such as superoxide anion (O₂ ^?) and H₂O₂ and activities of antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) were determined 6, 12, 24 and 48 h after inoculation. The measurements were done for ROS cytosolic fraction and enzymatic cytosolic or apoplastic fraction. Infection with Psp caused significant increase in ROS levels since the beginning of experiment. Activity of the apoplastic enzymes also increased remarkably at the beginning of experiment in contrast to the cytosolic ones. Cytosolic SOD and guaiacol peroxidase (GPOD) activities achieved the maximum values 48 h after treatment. Additional forms of the examined enzymes after specific Psp infection were identified; however, they were not present after single Bc inoculation. Subsequent Bc infection resulted only in changes of H₂O₂ and SOD that occurred to be especially important during plant–pathogen interaction. Cultivar Korona of common bean is considered to be resistant to Psp and mobilises its system upon infection with these bacteria. We put forward a hypothesis that the extent of defence reaction was so great that subsequent infection did not trigger significant additional response.     

Elevation of Defense Network in Chilli Against <Emphasis Type="Italic">Colletotrichum capsici</Emphasis> by Phyllospheric <Emphasis Type="Italic">Trichoderma</Emphasis> Strain

Biocontrol strategies have been mainly focused on proposing the use of biocontrol agents (BCAs) isolated from the rhizospheric region of the plant for protection against phytopathogens. The present study evaluates the effectiveness of phyllospheric Trichoderma isolates in elevating the defense responses in chilli against Colletotrichum capsici infection and comparing its efficiency to the conventionally recommended rhizospheric Trichoderma strains. The elicitation of the defense network in the plants was analyzed using biochemical assays for important enzymes, that is, PAL, PO, PPO, TPC, SOD along with the total protein level in challenged plants over untreated and unchallenged control plants. The results recorded 2.1, 5.18, 3, 0.67, and 0.5-fold increases in TPC, PAL, PO, PPO, and total protein content in BHUF4 (phyllopsheric Trichoderma isolate)-treated plants when compared to control plants under C. capsici challenge. This was at par with the increment recorded in T16A (rhizospheric Trichoderma isolate)-treated chilli plants. The increment in growth parameters was also recorded after treatment with the isolated Trichoderma strains. Interestingly, the phyllospheric isolate (BHUF4) treatment recorded comparable growth promotion in chilli plants recording 36, 62, and 60 % increases in one of the major parameters of plant growth, that is, root length, no. of leaves, and dry weight, respectively. This study proposes the use of combined application of both rhizospheric as well as phyllospheric Trichoderma isolates for better and all around protection of plants against foliar as well as soil phytopathogens. This would be a novel approach in biological control strategy for better management of anthracnose disease of chilli.