Expression of OsDREB2A in Transgenic Tomato Improves Drought Tolerance

Dehydration responsive element binding (DREB) are important regulatory molecules which have a crucial role in abiotic stress tolerance. The productivity of tomato, as a drought-sensitive crop, is highly restricted by drought stress. The current study aimed at introducing the OsDERB2A gene into two tomato genotypes via Agrobacterium-mediated transformation system. Cotyledonary explants were pre-cultured for two days with Agrobacterium strain LBA4404 harboring pCAMBIA1301 with OsDREB2A driven by the constitutive promoter CaMV35S for transformation. Shoots were directly regenerated on MS medium containing 1 mg l-1 zeatin and 1 mg l-1 BAP, and in presence of 30 mg l-1 hygromycin as selective agent. Only eight weeks were needed to regenerate transgenic tomato using this protocol. An OD600 of 0.4 resulted in 64.3-76.9% transformation efficiency. Stable integration and expression of the OsDREB2A gene were confirmed in transgenic tomato using PCR and RT-PCR analyses, and drought tolerance of T0 transgenic lines was confirmed by leaf disc assay in 300 mM mannitol. The superior biomass, photosynthetic pigments, free soluble sugars and proline accumulation of OsDREB2A transgenic lines over wild type in response to mannitol-stress revealed their enhanced drought tolerance and indicated that the constitutive expression of OsDREB2A might modulate the expression of other drought responsive genes.

A Leaf Disc Assay for Evaluating the Response of Tea (Camellia sinensis) to PEG-Induced Osmotic Stress and Protective Effects of Azoxystrobin against Drought

Tea (Camellia sinensis), a globally cultivated beverage crop, is sensitive to drought, which can have an adverse effect on the yield and quality of tea. Azoxystrobin (AZ) is one kind of fungicide considered as an agent to relieve damage caused by stress. Initially, the response of tea plant to osmotic-gradient stress was evaluated using leaf disc assays with PEG-induced osmotic stress. The decline of the maximum quantum yield of PSII (Fv/Fm), actual photosynthetic efficiency of PS II (Y(II)), total chlorophylls, carotenoids, DPPH radical scavenging capacity, reducing power, total phenols, and the increase in MDA was observed in leaf discs treated with a gradient of PEG solutions (22.8, 33.2, 41.1% PEG, and blank). These results revealed that efficiency of photosystem II (PSII), photosynthetic pigments, and antioxidant ability in leaf discs were inhibited with an aggravated lipid peroxidation under PEG-induced osmotic stress, and indicated leaf disc assay with moderate PEG iso-osmotic condition would reflect a portion of tea plant response to drought stress. Therefore, the protective effect of AZ (0.125 and 1.25 g a.i. L−1) on tea plants suffering from drought was evaluated using leaf disc assays with 22.8% PEG iso-osmotic condition. Pretreatment of AZ (0.125 a.i. g L−1) reversed Fv/Fm, Y(II), DPPH radical scavenging capacity, and reducing power with reduced MDA in PEG-treated leaf discs, but photosynthetic pigments, total phenols, and ascorbate peroxidase activity were irresponsive to AZ. An Alleviated physiological damage in tea leaf with AZ applying was preliminarily revealed in this study. A Rapid screening of agents for tea plants against drought was developed to assist in the selection of protective agents.

A Non-destructive Leaf Disc Assay for Rapid Diagnosis of Weed Resistance to Multiple Herbicides

Weed resistance survey that monitors the spread of resistant weeds has been mainly conducted through time-consuming, labor-intensive and destructive greenhouse herbicide screens. As an alternative, we here introduce a non-destructive leaf disc assay based on chlorophyll fluorescence (Fv/Fm values which measure photosynthesis efficiency) allowing the detection of resistance to both systemic and contact herbicides within ˜48h. Current study validated the assay on detecting resistance to fomesafen, glyphosate, and dicamba in Palmer amaranth (Amaranthus palmeri S. Watson), waterhemp [Amaranthus tuberculatus (Moq.) Sauer], kochia [Bassia scoparia (L.) A.J. Scott] and goosegrass [Eleusine indica (L.) Gaertn.]. Negative correlation between the Fv/Fm values and the spray injury levels was observed in all herbicide-weed combinations at the discriminating doses, except for glyphosate in Amaranthus. The correlation coefficients were -0.41 for fomesafen (10µM, p<0.0001) in Amaranthus, -0.92 for glyphosate in E. indica (250µM, p<0.0001), and -0.44 for dicamba in B. scoparia (800 µM, p=0.0023), respectively. At the population level, the assay clearly separated susceptible from highly resistant populations. However, the assay showed lower sensitivity in distinguishing populations of different resistance levels or separating low resistance from susceptible populations. At the individual plant level, results from the leaf disc assay and whole plant spray tests were concordant in 85.5%, 92.3%, and 71.7% of the plants tested for fomesafen-Amaranthus, glyphosate-Eleusine and dicamba-Bassia, respectively. The assay yielded 1-15% false positive and 6-13% false negative results across herbicides. The current study demonstrated that the leaf disc assay is a useful tool to identify weed resistance. Optimization is needed to improve its sensitivities and expand its usage to more diverse herbicide-weed species combinations.

Morphological, molecular and resistance responses to soft-rot disease variability among plantlets of Phalaenopsis amabilis regenerated from irradiated protocorms

Putri HA, Purwito A, Sudarsono, Sukma D. 2021. Morphological, molecular and resistance responses to soft-rot disease variability among plantlets of Phalaenopsis amabilis regenerated from irradiated protocorms. Biodiversitas 22: 1077-1090. Phalaenopsis amabilis (L.) Blume is a prominent donor for the white petal and sepal trait in Phalaenopsis breeding. However, it has an undesirable character, such as susceptible to soft-rot disease. Therefore, developing soft-rot resistance mutants through gamma irradiation could be explored. This study aimed to evaluate the variability of plantlets regenerated from irradiated and non-irradiated protocorms using morphology, stomatal size and molecular markers and to test responses of the plantlets against soft-rot disease. The plantlets were regenerated from irradiated (5, 10, 15 or 20 Gy) and non-irradiated protocorms. The results showed that P. amabilis plantlet variants were successfully identified based on their leaf morphology and stomatal size variations. A few plantlets have low stomatal densities, large stomatal size, and high chloroplast numbers, which indicated they were polyploids. Leaf disc assay for soft-rot disease response grouped most of the plantlets into very susceptible or susceptible. Moreover, four soft-rot resistant plantlets regenerated from irradiated and non-irradiated protocorms were successfully identified. The resistant plantlets were identified after three consecutive periods of inoculations with pathogens causing soft-rot disease. The evaluation also confirmed nucleotide variation in the Pto gene isolated from different levels of plantlet variant resistance responses.

Isolasi fungi endofit kulit mentimun (Cucumis sativus L.) dan evaluasi aktivitas penghambatannya terhadap pertumbuhan Candida albicans ATCC 10231

Mentimun (Cucumis sativus L.) merupakan salah satu tanaman dalam famili Cucurbitaceae yang banyak dibudidayakan. Uji fitokimia ekstrak kulit mentimun menunjukan adanya kandungan senyawa aktif yang berpotensi sebagai antifungi. Fungi endofit mampu memproduksi senyawa aktif yang mirip dengan senyawa yang dihasilkan oleh inangnya. Penelitian ini bertujuan untuk mengisolasi fungi endofit dari kulit mentimun dan menguji aktivitasnya sebagai antifungi terhadap Candida albicans. Fungi endofit diisolasi dari kulit mentimun varietas Baby. Isolat fungi endofit yang telah dipurifikasi kemudian dikarakterisasi secara morfologi dan diuji aktivitas antifunginya dengan metode paper disc assay. Selanjutnya dilakukan uji kualitatif untuk mendeteksi kandungan senyawa aktif antifungi dalam ekstrak isolat fungi endofit. Hasil penelitian menunjukkan bahwa terdapat 5 jenis isolat fungi endofit pada kulit mentimun yaitu AU1, AU2, AT3, BT1, BP2. Identifikasi secara morfologi menunjukkan bahwa isolat AU1 dan BT1 termasuk pada Penicillium, isolat AU2 dan AT3 sebagai anggota genus Cladosporium, dan isolat BP2 teridentifikasi sebagai Aspergillus. Kelima isolat mampu menghambat pertumbuhan Candida albicans. Hasil uji terhadap supernatan masing-masing isolat mengungkap bahwa isolat AU1 dan AT3 terdeteksi memiliki kandungan senyawa flavonoid, isolat BP2 menghasilkan alkaloid dan flavonoid, sedangkan isolat BT1 menghasilkan flavonoid, saponin dan tanin, serta isolat AU2 menghasilkan steroid, flavonoid, saponin dan tanin. Senyawa - senyawa metabolit tersebut berpotensi sebagai antifungi.

Identification and Characterization of Antagonism Band of Secondary Metabolite from T. asperellum MK045610 against F. oxysporum f. sp. ciceri and F. oxysporum f. sp. lycopersici based on HPTLC and GC-MS

The present investigation was carried out on identification and characterization of an antagonism band of extract secondary metabolites from Trichoderma asperellum MK045610 against Fusarium oxysporum f. sp. ciceri and Fusarium oxysporum f. sp. lycopersici. In this experiment, analysis was performed by High Performance Thin Layer Chromatography (HPTLC), paper disc assay, gas chromatography-Mass spectrometry (GC-MS). Firstly, extract crude secondary metabolites were used for partial purification based on HPTLC. Secondly, the paper disc assay method was used for the determination of antifungal property on PDA plate from the partial purified compound. Thirdly, GC-MS was used for identification of partial purified compound based on peaks. Identified compounds are named as Phenol, 3, 5-bis (1,1-dimethylethyl), Pentadecanoic Acid, 14-methyl, methyl ester, Benzenepropanoic acid, 3,5,-bis (1,1-dimethyl ethyl)-4-hydroxy-methyl ester and represented to antifungal property . Conclusively, Secondary metabolite of Trichoderma asperellum MK045610 has a significant role in radial growth inhibition of Fusarium oxysporum f. sp. ciceri and Fusarium oxysporum f. sp. lycopersici.

Occurrence and feeding preference of Diabrotica speciosa Germar and Cerotoma arcuata (Olivier) for different cultivars of cowpea Vigna unguiculata (Linnaeus) Walpers

Chrysomelid pests are not only known as leaf-feeding in beans but as vectors of viruses too. Chemical control is most commonly used but has high economic and environmental costs. This study aimed to report the occurrence of chrysomelid pests on cowpea area in the State of Tocantins and compare the feeding preference of the species collected in different cowpea cultivars. Weekly monitoring was carried out in cowpea stand at the experimental station of the Federal University of Tocantins, in Gurupi-TO. Insect feeding preference was evaluated under laboratory conditions (25 ± 2 °C, 60 ± 5% R.H.). A total of 1,542 adults of two species of chrysomelid pests was recorded: Cerotoma arcuata (Olivier) presented 71.49% and Diabrotica speciosa Germar, 28.51%. Leaf discs of the cultivars BRS "Tumucumaque"," BRS "Nova Era", BRS "Sempre Verde", and "Pingo de Ouro", were offered to adult beetles. It was observed a significant difference in no-choice leaf disc assays and four-choice leaf disc assays for the D. speciosa species, being BRS "Tumucumaque" the most consumed. In the no-choice leaf disc assay "Pingo de Ouro" was less consumed and showed non-preference type resistance. In the four-choice leaf disc assay, BRS Sempre Verde was more resistant to D. speciosa consumption. In both tests, C. arcuata did not show a significant difference among the tested cultivars, but mathematically, BRS "Nova Era" was the most consumed, while "Pingo de Ouro" the lowest consumed and showed non-preferred resistance in both tests. Cerotoma arcuata stood out as greater defoliant than D. speciosa in all cultivars tested.Ocorrência e preferência alimentar de Diabrotica speciosa Germar e Cerotoma arcuata (Olivier) por diferentes cultivares de feijão caupi Vigna unguiculata (Linnaeus) WalpersResumo. Crisomelídeos pragas não são apenas desfolhadores do feijoeiro, mas atuam também como vetores de viroses. O método de controle mais utilizado é o químico, o que acarreta altos custos econômicos e ambientais. Este trabalho objetivou registrar a ocorrência de crisomelídeos pragas em cultivo de feijão-caupi no estado do Tocantins, bem como comparar a preferência alimentar das espécies coletadas por diferentes cultivares. Foi realizado monitoramento semanal na área de feijão-caupi da estação experimental da Universidade Federal do Tocantins em Gurupi-TO. Foram registrados 1.542 adultos de duas espécies de crisomelídeos pragas: Cerotoma arcuata (Olivier)apresentou 71,49% e Diabrotica speciosa Germar, 28,51%. Em condições de laboratório, discos foliares das cultivares BRS "Tumucumaque", BRS "Nova Era", BRS "Sempre Verde" e "Pingo de Ouro" foram oferecidos a insetos adultos para testar sua preferência alimentar. Foi observada diferença significativa nos ensaios sem chance e com chance de escolha para espécie de D. speciosa, sendo o BRS "Tumucumaque" o mais consumido. Nos ensaios sem chance de escolha, "Pingo de Ouro" foi menos consumido e apresentou resistência do tipo não preferência. Nos ensaios com chance de escolha, o cultivar BRS "Sempre Verde" foi mais resistente ao consumo de D. speciosa. Em ambos os testes, C. arcuata não apresentou diferença significativa na preferência pelos quatro cultivares, embora BRS "Nova Era" tenha sido mais consumido em valores absolutos, enquanto "Pingo de Ouro" foi menos desfolhado apresentando resistência de tipo não preferência nos dois testes. Cerotoma arcuata destacou-se como maior desfolhador do que D. speciosa em todas os cultivares testados.

A detached petal disc assay and virus-induced gene silencing facilitate the study of Botrytis cinerea resistance in rose flowers

Fresh-cut roses (Rosa hybrida) are one of the most important ornamental crops worldwide, with annual trade in the billions of dollars. Gray mold disease caused by the pathogen Botrytis cinerea is the most serious fungal threat to cut roses, causing extensive postharvest losses. In this study, we optimized a detached petal disc assay (DPDA) for artificial B. cinerea inoculation and quantification of disease symptoms in rose petals. Furthermore, as the identification of rose genes involved in B. cinerea resistance could provide useful genetic and genomic resources, we devised a virus-induced gene silencing (VIGS) procedure for the functional analysis of B. cinerea resistance genes in rose petals. We used RhPR10.1 as a reporter of silencing efficiency and found that the rose cultivar ‘Samantha’ showed the greatest decrease in RhPR10.1 expression among the cultivars tested. To determine whether jasmonic acid and ethylene are required for B. cinerea resistance in rose petals, we used VIGS to silence the expression of RhLOX5 and RhEIN3 (encoding a jasmonic acid biosynthesis pathway protein and an ethylene regulatory protein, respectively) and found that petal susceptibility to B. cinerea was affected. Finally, a VIGS screen of B. cinerea-induced rose transcription factors demonstrated the potential benefits of this method for the high-throughput identification of gene function in B. cinerea resistance. Collectively, our data show that the combination of the DPDA and VIGS is a reliable and high-throughput method for studying B. cinerea resistance in rose.