Development of Gene-Based InDel Markers on Putative Drought Stress-Responsive Genes and Genetic Diversity of Durian (Durio zibethinus).

Insertion-deletion (InDel) markers are co-dominant, relatively abundant and practical for agarose gel genotyping. InDel polymorphism usually affects gene functions. Nucleotide sequences of durian (Durio zibethinus) are available, but InDel makers have not been well established. This study aimed to develop drought-related gene-based InDel markers for durian through bioinformatic analysis of RNA-Seq datasets. The polymorphism of the markers was verified in 24 durian genotypes local to Thailand. Bioinformatic analysis indicated 496 InDel loci having lengths more than 9 bp. To evaluate these InDel markers, 15 InDel loci were selected. Nine markers were successfully amplified a clear polymorphic band pattern on 2% agarose gel. The polymorphic information content (PIC) of these nine markers ranged from 0.1103 to 0.5808. The genetic distance between the 24 genotypes ranged from 0.222 to 0.889. The phylogeny based on the nine InDel loci distinguished the 24 genotypes and divided samples into four groups. This set of gene-based InDel markers on putative drought-responsive genes will be useful for genetic studies.

Genotypic variation in 9-Cis-Epoxycarotenoid Dioxygenase3 gene expression and abscisic acid accumulation in relation to drought tolerance of Hevea brasiliensis.

Abscisic acid (ABA) is a stress-related plant hormone, which is reported to confer drought tolerance. A key enzyme in ABA biosynthesis is 9-cis-epoxycarotenoid dioxygenase. In this study, changes in morphological, physiological response, HbNCED3, and ABA accumulation of RRIM 623 and PB 5/51 rubber clones were observed at different time points of water deficit conditions (0, 3, 5, 7, and 9 days of withholding water). During water deficit, the relative water content (RWC), photosynthetic rate (Pn), and stomatal conductance (Gs) decreased, whereas the electro leakage (EL) increased. The magnitudes of the changes in these parameters were greater for PB 5/51 than for RRIM 623. Therefore, RRIM 623 was designated as representative of drought-tolerant clone and PB 5/51 as a drought-sensitive clone. The HbNCED3 transcription level of RRIM 623 showed lower expression compared with that of PB 5/51, which corresponded to the accumulation of ABA. RRIM 623 accumulated less ABA than PB 5/51. The ABA in RRIM 623 gradually increased, especially on the 7th day of withholding water, whereas that in PB 5/51 rapidly increased during the early periods of drought conditions. Additionally, the sensitivity of stomatal response to ABA showed that RRIM 623 had a higher sensitivity than PB 5/51. These results demonstrate that the drought-tolerant rubber clone, RRIM 623, was characterized by lower ABA accumulation during drought stress than the drought-sensitive clone, PB 5/51. The drought tolerance mechanism of the RRIM 623 might be associated with stomatal sensitivity to ABA accumulation under drought stress.

Antioxidant enzymatic activities and gene expression associated with heat tolerance in the stems and roots of two cucurbit species ("Cucurbita maxima" and "Cucurbita moschata") and their interspecific inbred line "Maxchata".

The elucidation of heat tolerance mechanisms is required to combat the challenges of global warming. This study aimed to determine the antioxidant enzyme responses to heat stress, at the enzymatic activity and gene expression levels, and to investigate the antioxidative alterations associated with heat tolerance in the stems and roots of squashes using three genotypes differing in heat tolerance. Plants of heat-tolerant "C. moschata", thermolabile "C. maxima" and moderately heat-tolerant interspecific inbred line "Maxchata" genotypes were exposed to moderate (37 °C) and severe (42 °C) heat shocks. "C. moschata" exhibited comparatively little oxidative damage, with the lowest hydrogen peroxide (H2O2), superoxide (O2(-)) and malondialdehyde (MDA) contents in the roots compared to stems, followed by "Maxchata". The enzyme activities of superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT) and peroxidase (POD) were found to be increased with heat stress in tolerant genotypes. The significant inductions of FeSOD, MnSOD, APX2, CAT1 and CAT3 isoforms in tolerant genotypes suggested their participation in heat tolerance. The differential isoform patterns of SOD, APX and CAT between stems and roots also indicated their tissue specificity. Furthermore, despite the sequence similarity of the studied antioxidant genes among "C. maxima" and "Maxchata", most of these genes were highly induced under heat stress in "Maxchata", which contributed to its heat tolerance. This phenomenon also indicated the involvement of other unknown genetic and/or epigenetic factors in controlling the expression of these antioxidant genes in squashes, which demands further exploration.

Overcoming Encouragement of Dragon Fruit Plant (Hylocereus undatus) against Stem Brown Spot Disease Caused by Neoscytalidium dimidiatum Using Bacillus subtilis Combined with Sodium Bicarbonate.

The use of the supernatant from a Bacillus subtilis culture mixed with sodium bicarbonate was explored as a means of controlling stem brown spot disease in dragon fruit plants. In in vitro experiments, the B. subtilis supernatant used with sodium bicarbonate showed a strong inhibition effect on the growth of the fungus, Neoscytalidium dimidiatum, the agent causing stem brown spot disease and was notably effective in preventing fungal invasion of dragon fruit plant. This combination not only directly suppressed the growth of N. dimidiatum, but also indirectly affected the development of the disease by eliciting the dragon-fruit plant's defense response. Substantial levels of the pathogenesis-related proteins, chitinase and glucanase, and the phenylpropanoid biosynthetic pathway enzymes, peroxidase and phenyl alanine ammonia-lyase, were triggered. Significant lignin deposition was also detected in treated cladodes of injured dragon fruit plants in in vivo experiments. In summary, B. subtilis supernatant combined with sodium bicarbonate protected dragon fruit plant loss through stem brown spot disease during plant development in the field through pathogenic fungal inhibition and the induction of defense response mechanisms.

Bioaugmentation coupled with phytoremediation for the removal of phenolic compounds and color from treated palm oil mill effluent.

The potential for coupling bioaugmentation with phytoremediation to simultaneously treat and utilize treated palm oil mill effluent (TPOME) in animal feed production was determined from a reduction in phenolic compounds and color in soil leachates, as well as from an increased yield of pasture grass. Two phenol-degrading bacteria-Methylobacterium sp. NP3 and Acinetobacter sp. PK1-were inoculated into the Brachiaria humidicola rhizosphere before the application of TPOME. A pot study showed that the soil with both grass and inoculated bacteria had the highest dephenolization and decolorization efficiencies, with a maximum capability of removing 70% from 587 mg total phenolic compounds added and 73% from 4438 color units during ten TPOME application cycles. The results corresponded to increases in the number of phenol-degrading bacteria and the grass yield. In a field study, this treatment was able to remove 46% from 21,453 mg total phenolic compounds added, with a maximum color removal efficiency of 52% from 5105 color units, while the uninoculated plots removed about 24-39% and 29-46% of phenolic compounds and color, respectively. The lower treatment performance was probably due to the increased TPOME concentrations. Based on the amounts of phenolic compounds, protein, and crude fiber in the grass biomass, the inoculated TPOME-treated grass had a satisfactory nutritional quality and digestibility for use as animal feed.

Cloning and Expression Analysis of HbPR-1b and HbPR-3 in Hevea brasiliensis During Inoculation with Rigidoporus microporus.

BACKGROUND AND OBJECTIVE: Pathogenesis-related (PR) proteins are dramatically accumulated after pathogen infection. Innate defense response through increasing PR-proteins is important for rubber rootstock selection that is tolerant to the white root disease caused by Rigidoporus microporus. This study was aimed to investigate the expression levels of PR-1 and PR-3 genes in tolerant (PB5/51) and susceptible (BPM24 and RRIM600) rubber clones after R. microporus infection. MATERIALS AND METHODS: The mRNA of HbPR-1b and HbPR-3 was isolated and characterized from rubber leaves. Gene expression levels of HbPR-1b and HbPR-3 were compared among three rubber clones (PB5/51, BPM24 and RRIM600) after R. microporus infection at 0, 12, 24, 48, 72 and 96 h using quantitative real-time PCR. The relative transcript abundances between inoculated and control plants were compared using the means of gene expression between time points and by Tukey's HSD test. A probability value (p<0.05) was used to determine the significance of difference between time points. RESULTS: The open reading frame of HbPR-1b is 492 bp with deduced 163 amino acid residues and the phylogenetic analysis showed it shared significant evolutionary history and clustering into group I of PR-protein. Moreover, the partial HbPR-3 was isolated with 390 bp. Gene expression levels of HbPR-1b and HbPR-3 showed marked differences in both transcripts depending on the rubber clones. Two genes demonstrated up-regulation of both tolerance and susceptibility in response to attack by R. microporus. The highest expression levels were found in seedlings of PB5/51 after inoculation. In RRIM600, low expression levels of HbPR-1b and HbPR-3 were initially observed but gradually increased at 24 h post inoculation. The transcription profile of HbPR-1b was stable expression in BPM24. CONCLUSION: The results demonstrated that the level ofHbPR-1b and HbPR-3 transcription can distinguish between tolerant and susceptible clones. The candidate defense genes to the white root disease were observed in PB5/51 seedlings, particularly HbPR-1b.

Carotenoid accumulation and carotenogenic gene expression during fruit development in novel interspecific inbred squash lines and their parents.

Carotenoid levels and composition during squash fruit development were compared in Cucurbita moschata , Cucurbita maxima , and two lines of their interspecific inbred lines, namely, Maxchata1 and Maxchata2. Eight genes associated with carotenoid biosynthesis were analyzed by quantitative RT-PCR. The two squash species and their interspecific inbred lines exhibited different qualitative and quantitative carotenoid profiles and regulatory mechanisms. C. moschata had the lowest total carotenoid content and mainly accumulated α-carotene and ß-carotene, as expected in a fruit with pale-orange flesh. Low carotenoid content in this species was probably due to the comparatively low expression of all genes investigated, especially PSY1 gene, compared to the other squashes. The predominant carotenoids in C. maxima were violaxanthin and lutein, which produced a corresponding yellow flesh color in mature fruit. The relationship between the expression of the CHYB and ZEP genes may result in almost equal concentrations of violaxanthin and lutein in C. maxima at fruit ripening. In contrast, their interspecific inbred lines principally accumulated lutein and ß-carotene, leading to orange flesh color. The PSY1 gene exhibited higher expression levels at earlier stages of fruit development in the Maxchata lines, potentially triggering the increased carotenoid accumulation seen in these fruits. Likewise, the higher transcription level of CHYB gene observed in the two interspecific inbred lines might be correlated with high lutein in these hybrids. However, this study could not explain the observed ß-carotene accumulation on the basis of gene expression.