Identification of the Pathogen Causing Stem Blight Disease on Chili in Sindangjaya Village, Cipanas, Cianjur, West Java Based on Morphological and Molecular Analyses

<p>Chili (Capsicum annuum L.) is a vegetable commodity with high economic value which is widely cultivated by farmers in Indonesia. One of the obstacles faced in chili cultivation is stem rot disease. This study aimed to identify the pathogens that caused stem rot in chili plants obtained from one location in Sindangjaya Village, Cipanas District, Cianjur Regency, West Java Province based on morphological and molecular analyses. Pathogen identification was performed with morphological and molecular approaches. The morphological characters observed included colony shape, sporangium diameter, and mating type. The pathogenicity of the isolates was assayed by inoculating chili stems aged 40 days. Molecular identification was carried out using two pairs of primers for ITS regions and TEF-1 gene. Based on the results of morphological and molecular identification, as well as pathogenicity tests, it was confirmed that Phytophthora capsici pathogen was the causal agent of stem rot in chili plants collected from Sindangjaya Village. Further study is needed to determine the spread of the disease, damage, and yield loss caused by stem rot disease, as well as how to prevent and control the disease.</p>

Resistance Analysis of CRISPR/Cas9 Genome-Edited Chili M2 Mutant Lines against Pepper Yellow Leaf Curl Viral Disease

<p>Pepper yellow leaf curl virus (PepYLCV) infection transmitted by silverleaf whitefly (Bemisia tabaci [Gennadius]) can decrease chili pepper yield up to 100%. At this moment, there is no chili pepper variety resistant to PepYLCV available. Genome editing approach through CRISPR/Cas9 is an effort to develop variety resistance to the viral infection. The purpose of this study was to obtain M2 lines developed by CRISPR/Cas9 system on proliferating cell nuclear antigen (PCNA) gene for resistance to PepYLCV. A total of four M2 lines (C47-7, L84-2, L84-23, and L120-19) consisting of 60 chili plants were tested for their resistance to PepYLCV. PCR analysis was performed to detect the presence (infection) of the virus. The results showed that a total of 35 plants derived from the four lines were resistant to PepYLCV. They consisted of 7 plants from C47-7 line, 11 plants from L84-2 line, 9 plants from L84-23 line, and 8 plants from L120-19 line. PCR analysis confirmed that the resistant plants obtained from this study were negatively infected by the virus. Since not all tested plants were resistant to virus infection, the PCNA gene allele in these resistant lines were most likely heterozigotes. Sequencing of PCNA gene of the resistant lines is needed to confirm that the resistance phenotypes obtained was due to mutation of the gene. Therefore, further selection needs to be performed to obtain stable and PepYLCV-resistant lines.</p>

Utilization of Molecular Markers for Rice Breeding

Rice is the staple food for more than half of the world's population. Rice production in 2050 must increase by at least 50% to keep up with the population growth. Efforts to increase rice production continue using various strategies. Breeders apply multiple approaches including application of molecular markers in developing varieties better than the previous ones. Since the discovery of the restriction fragment length polymorphism (RFLP) markers in 1980s and the development of polymerase chain reaction (PCR) method, many types of molecular markers have been developed and applied to various crops including rice. Various molecular approaches to map genetic loci associated with rice superior traits were conducted. The mapped loci are very useful for rice breeding purposes. This paper reports the results of mapping and breeding economically important traits in rice, mainly those related to abiotic stresses, agronomic traits, yield, and yield quality. These included characters of semidwarf stature, aromatic grain, high yield potential, eating quality, higher Zn and Fe grain, more tolerant to abiotic stresses, such as salinity, drought, phosphate deficiency, Al toxicity and Fe toxicity, submergence, as well as early maturity character. The mapped characters can be transferred using marker-assisted backcrossing (MABC) method into cultivated rice genotypes well-adopted by farmers. Several countries including Indonesia have benefited from this breeding method, and Indonesia have released several rice varieties developed through MABC. These include rice varieties such as Code, Angke, Inpari 30, Inpari Blas, Inpari HDB, Bio Patenggang Agritan, and Bioni 63 Ciherang Agritan

Genetic Diversity Analysis of Indonesian Pigmented Rice Accessions Based on Functional Molecular Markers

Pigmented rice is functional staple food that becomes popular because of its anthocyanin content which is beneficial for health. Studies on the diversity of the local variety of Indonesian pigmented rice accessions have been carried out, but are still limited to one region of germplasm origin. This study aimed to analyze the genetic diversity of local varieties of pigmented rice collections of the IAARD-ICABIOGRAD Gene Bank. A total of 93 pigmented rice accessions from 16 provinces in Indonesia were analyzed using 15 functional molecular markers of SSR, STS, and indel. The total alleles detected were 115 with an average per locus of genetic diversity value of 0.71. There were five markers with PIC values &gt;0.75, i.e. RM167, RM223, R8M33, R10M10, and GBSS1. The accessions were divided into two main groups based on their pericarp color. It is necessary to analyze the physicochemical content of the local rice accessions to complement the existing diversity information and identify potential pigmented rice accessions with high palatability.

Development of Transgenic Sugarcane Resistant to Stem Borer by Transforming cry1Ab-cry1Ac Fusion Gene through Agrobacterium tumefaciens Transformation Method

<p>Bulu Lawang (BL) is a sugarcane variety preferred by farmers in Indonesia due to its high yield, but this cultivar is susceptible to shoot and stem borer insect pests. Genetic engineering using cry1Ab and cry1Ac fusion gene is an effort to generate BL varieties resistant to the insect pests. This study aimed to 1) transform T-DNA containing cry1Ab-cry1Ac fusion gene into sugarcane genome by using Agrobacterium tumefaciens method, 2) obtain selection media composition of callus transformants, and 3) obtain transformation efficiency comparison of A. tumefaciens strains EHA105 and GV3101. The research was conducted at the Laboratory of Cell and Tissue Biology, the Laboratory of Molecular Biology, and the greenhouse of the Indonesian Center for Agricultural Biotechnology and Genetic Resources Research and Development, Bogor from March to August 2019. Research activities consisted of four parts namely 1) callus induction and Agrobacterium culture preparation containing pCambia5300-cry1Ab-cry1Ac ///// pCambia-5300_OaRbcS-prom-cTP-cry1Ab-cry1Ac plasmid, 2) callus incubation in cocultivation and resting media, 3) selection and differentiation of shoots on regeneration media, and 4) molecular analysis using PCR method. Results showed that the composition of media, both for selection and regeneration processes of putative plant transformants, was the key to the success of this experiment. A. tumefaciens strain EHA105 resulted in higher transformation efficiency (11.1%) compared to that of strain GV3101 (9.0%). Molecular analysis showed that cryIAb-cryIAc fusion gene was successfully inserted into the sugarcane genome suggesting that the transgenic plant containing cry1Ab-cry1Ac fusion gene was obtained. The putative transgenic plants need further assay through bioassay tests to verify its resistance phenotype to the insect pests.</p>

Improving Cassava Productivity by Soil Bioaugmentation with Phosphate-Solubilizing Actinomycetes and Fungi

<p>Cassava is one of the most important food commodities, but its cultivation technique must be improved, especially the technology in increasing soil fertility. Lack of phosphate (P), one of the major biological nutrients in soil, can reduce<br />agricultural production. Some P-solubilizing microbes can play an important role in increasing the availability of P for plants. The purpose of this study was to evaluate the ability of P-solubilizing microbes isolated from cassava rhizosphere in improving the growth of cassava after soil bioaugmentation with the formula of selected microbes. A total of 50 isolates were successfully isolated from cassava plant rhizosphere collected from several locations in South Sulawesi. In vitro screening on Pikovskaya agar media resulted in six Actinomycetes and six fungal isolates with the best P hydrolysis capacity. One Actinomycetes isolate<br />(Streptomyces sp. A-SDR01) and one fungal isolate (Penicillium sp. F-SKG17) with nonantagonistic effect to each other based on in vitro test were able to improve the vegetative growth of cassava under in planta test. Combination of both isolates in a gum arabic formulation increased plant height and productivity compared to untreated plants when applied as soil bioaugmentation in limited field trials at four locations in South Sulawesi. Therefore, application of P-solubilizing microbes that possess soil bioaugmentation properties is recommended for increasing the growth of cassava plants and their use as<br />microbial biofertilizers should be extended to wider areas.</p>

Editing of PCNA Gene by CRISPR/Cas9 Technology to Improve the Red Chili Resistance to Yellow Leaf Curl Disease

<p>Yellow leaf curl disease caused by Pepper yellow leaf curl virus (PepYLCV), member of geminiviruses group, is responsible for heavy yield losses for chili pepper production. Resistant genes which can cause immunity to the disease have not been found in germplasm collection. The aim of the research was to edit proliferating cell nuclear antigen (PCNA) gene by using CRISPR/Cas9 technology for developing plant resistance against geminivirus in chili pepper. A CRISPR/Cas9 plasmid cassette construct harboring the guide RNA of PCNA gene was constructed by Golden Gate cloning strategy. The construct was then introduced into chili genome via in planta method using Agrobacterium tumefaciens EHA105. The transformed plants were bioassayed by virus inoculation and confirmed using PCR and DNA sequencing to identify a mutagenesis event in PCNA gene target. The results showed that CRISPR/Cas9 plasmid cassette harboring gRNA of PCNA gene was successfully constructed. In planta transformation using A. tumefaciens vector harboring CRISPR/Cas9-gRNA PCNA construct resulted in 307 and 193 transformed plants from chili var. Lingga and Ciko, respectively. Bioassay by using virus inoculation to the transformed plants obtained 6 and 14 lines of Lingga and Ciko, respectively, which were resistant to geminivirus (no symptom observed). The resistant lines of chili pepper var. Lingga and Ciko were mutated in PCNA gene with one base insertion or deletion mutation types. These results exhibit that the CRISPR/Cas9 genome editing can be used to induce mutant of PCNA gene in chili pepper. Further investigation is necessary to evaluate the selected chili lines resistant to PepYLCV infection.</p>

Exploration of IAA and HCN Producing Rhizobacteria from Cassava (Manihot esculenta Crantz) Rhizosphere

<p>Plant growth promoting rhizobacteria (PGPR) are a group of beneficial bacteria that live in rhizosphere. These bacteria can promote plant growth through several mechanisms, such as the ones produce indole-3-acetic acid (IAA) hormone and hydrogen cyanide (HCN), and act as biocontrol agents. The use of PGPR to promote plant growth has been known to be an environmentally friendly alternative approach. The aim of this study was to explore IAA and HCN producing rhizobacteria from cassava rhizosphere soil and identify the bacteria based on morphological and biochemical characters, hypersensitive reaction test, and the ability test to produce IAA and HCN. The results showed nine bacterial isolates suspected as Micrococcus sp. (six isolates), Neisseria sp. (two isolates), and Bacillus sp. (one isolate). All isolates were able to produce IAA in the concentration range of 50,63–135,00 µg/ml and 232,3–333,9 µg/ml at incubation time of 2 and 4 days, respectively. All isolates were able to produce HCN. In addition, the isolates did not show hypersensitivity reactions. Further study is needed to assess the isolate application for promoting plant growth as well as a biocontrol agent of plant pathogen.</p>

Isolation and Homology Analysis of Alanine Aminotransferase Gene of Barley, Foxtail Millet, Cucumber, and Tomato

<p>Overexpression of <em>alanine aminotransferase<strong> </strong></em>(<em>AlaAT</em>) gene can improve nitrogen use efficiency (NUE) in plants. The previous isolated <em>AlaAT</em> genes cannot be freely applied to generate NUE plants due to IPR restriction. Therefore, isolation of the gene from targeted monocot and dicot plants is necessary. The objectives of this study were to isolate <em>AlaAT</em> genes from barley, foxtail millet, cucumber, and tomato and analyze their homology to other isolated <em>AlaAT</em> genes in sequence databases (gene bank). Total RNA was isolated from roots of barley, foxtail millet, cucumber, and tomato, and then converted into cDNA using reverse transcription method. The cDNA was then cloned into <em>pGEM®-T Easy</em> plasmid and the verified clones were sequenced. The amino acid sequences were analyzed for their homologies using Clustal Omega software and phylogenetic tree was constructed. The results showed that the amino acids of <em>AlaAT</em> gene from barley was different from <em>AlaAT</em> genes of tomato and cucumber with homology level less than 80%. Phylogenetic tree predicted that <em>AlaAT</em> genes clustered into three groups with <em>AlaAT</em> genes of foxtail millet and barley clustered in one group together with other monocots in group I. <em>AlaAT</em> genes derived from dicots clustered into two groups<em> </em>in which<em> AlaAT</em> gene of tomato clustered in group II, while that of cucumber was in group III. The identity differences of <em>AlaAT</em> gene of tomato and that of cucumber as well as the estimates of the same enzymatic functions can open up enormous opportunities in genetic engineering research for the development of NUE rice.</p>

Genetic Diversity Analysis and Development of DNA Fingerprints of 20 Indonesian Local Chili Pepper Varieties Based on SSR Markers

<p>Chili pepper is one of the most valuable<em> </em>horticultural crops, widely cultivated in Indonesia. Analysis of its genetic diversity is needed to develop successful breeding programs of local varieties. Simple sequence repeat (SSR), a robust molecular marker used for genetic diversity analysis in plant species, offers potential, reliable DNA fingerprinting method to assess genetic variation and varietal identification of chili pepper. Fifteen SSR markers were used in this study to analyze the genetic diversity and develop profiling identification of DNA fingerprint of local chili pepper varieties. Twenty local and two improved varieties of three chili pepper species, consisting of 3, 1, and 18 varieties of <em>Capsicum </em><em>frutescens</em>, <em>C. chinense</em>, and <em>C. annuum</em>, respectively, were assessed for their SSR polymorphism. A total of 87 alleles was obtained from the polymorphism analysis with high alleles variation (2–16 alleles) with average total allele of 5.8 and average polymorphism information content (PIC) of 0.59 (0.34–0.83). Clustering and Principle Coordinate Analyses (PCoA) classified the varieties into two groups with coefficient of similarity of 0.65 indicating their high genetic variability. Most local varieties belonged to the same cluster and separated from the two improved varieties. Based on PIC values and dendrogram with selected markers, five SSR markers, i.e. EPMS441, EPMS331, EPMS335, GPMS194, and CaSSRBio1.1, were identified as SSR marker set for DNA fingerprinting purposes. SSR marker set used in this study was successful in developing the varietal identity of local chili pepper varieties, as indicated by unique code of each variety.</p>