DOMESTICATION AND FORMULATION OF RHIZOPODOPSIS JAVENSIS AS A TEMPEH STARTER

ABSTRACT Domestication of wild fungal strains involved in the manufacture of traditional fermented foods often occurs spontaneously. Rhizopodopsis javensis (Rh. javensis) is taxonomically close to Rhizopus. The wild strain Rhizopodopsis javensis has found in cool climates can be developed as a starter in tempeh production in temperate regions. Before formulating it as a tempeh starter, a wild strain of Rh. javensis needs to be domesticated in human-made niches. A wild strain of Rh. javensis was domesticated by subculture using rice flour media at optimum growth temperature and carried out every five days. The spore's density and viability and the starter's water content were used to determine its quality. The results showed that Rh. javensis grew optimally at 22 ℃. With seven-time subcultures using rice flour media, the domestication process did not change the Rh. javensis growth rate and colony appearance. The growth rate of Rh. javensis is relatively the same as that of commercial tempeh starter and pure R. microsporus var. oligosporus, at each optimal growth temperature. In the rice flour media as a carrier, Rh. javensis produces spore's density that is relatively the same as that of commercial tempeh starter but with lower spore's viability and higher water content. Therefore, Rh. javensis cannot be used as a starter to produce tempeh in the temperate region. The carrier material and drying processes still need to be modified to increase spore viability and improve the overall quality, including the starter's lifespan. Keywords: food fermentation, Rhizopus microsporus var. oligosporus, spore's viability, starter quality, wild strain ABSTRAK Domestikasi galur liar kapang yang terlibat dalam dalam pembuatan makanan fermentasi tradisional, sering terjadi secara spontan. Rhizopodopsis javensis (Rh. javensis) merupakan salah satu galur liar kapang yang memiliki hubungan taksonomi dekat dengan Rhizopus. Strain liar ini ditemukan di daerah beriklim sejuk, sehingga berpotensi untuk dikembangkan sebagai starter tempe untuk produksi di daerah beriklim sedang. Untuk mendapatkan kultur yang tumbuh subur di relung (niches) buatan manusia, strain liar Rh. javensis perlu didomestikasi terlebih dahulu. Penelitian ini bertujuan untuk mendomestikasi strain Rh. javensis liar yang dilanjutkan dengan memformulasikannya sebagai starter tempe. Domestikasi dilakukan dengan menumbuhkan strain liar Rh. javensis pada media tepung beras pada suhu pertumbuhan optimum dan diulangi setiap lima hari. Kerapatan dan viabilitas spora, serta kadar air starter digunakan sebagai penilaian keberhasilan starter. Hasil penelitian menemukan Rh. javensis tumbuh optimal pada suhu 22 ℃. Domestikasi dengan cara subkultur koloni Rh. javensis pada media tepung beras selama 7 kali tidak mengubah kecepatan pertumbuhan Rh. javensis dan penampakan koloni. Laju pertumbuhan Rh. javensis relatif sama dengan laju pertumbuhan starter tempe komersial dan R. microsporus var. oligosporus murni, pada suhu optimum pertumbuhan masing-masing. Formulasi tepung beras sebagai media pembawa starter Rh. javensis, menghasilkan kerapatan spora yang relatif sama dengan starter tempe komersial, namun viabilitas sporanya rendah dan kadar airnya tinggi. Starter Rh. javensis belum dapat digunakan untuk membuat tempe. Substrat dan proses pengeringan masih perlu dimodifikasi untuk meningkatkan viabilitas spora dan kualitas starter tempe secara keseluruhan, termasuk umur simpan starter. Keywords: fermentasi makanan, kualitas starter, Rhizopus microsporus var. oligosporus, strain liar, viabilitas spora

S. pombe wtf genes use dual transcriptional regulation and selective protein exclusion from spores to cause meiotic drive

Meiotic drivers bias gametogenesis to ensure their transmission into more than half the offspring of a heterozygote. In Schizosaccharomyces pombe, wtf meiotic drivers destroy the meiotic products (spores) that do not inherit the driver from a heterozygote, thereby reducing fertility. wtf drivers encode both a Wtf poison protein and a Wtf antidote protein using alternative transcriptional start sites. Here, we analyze how the expression and localization of the Wtf proteins are regulated to achieve drive. We show that transcriptional timing and selective protein exclusion from developing spores ensure that all spores are exposed to Wtf4 poison, but only the spores that inherit wtf4 receive a dose of Wtf4 antidote sufficient for survival. In addition, we show that the Mei4 transcription factor, a master regulator of meiosis, controls the expression of the wtf4 poison transcript. This dual transcriptional regulation, which includes the use of a critical meiotic transcription factor, likely complicates the universal suppression of wtf genes without concomitantly disrupting spore viability. We propose that these features contribute to the evolutionary success of the wtf drivers.

Genome-wide identification of sexual-reproduction genes in fission yeast via transposon-insertion sequencing

Many genes required for sexual reproduction remain to be identified. Moreover, many of the genes that are known have been characterized in distinct experiments using different conditions, which complicates understanding the relative contributions of genes to sex. To address these challenges, we developed an assay in Schizosaccharomyces pombe that couples transposon mutagenesis with high-throughput sequencing (TN-seq) to quantitatively measure the fitness contribution of nonessential genes across the genome to sexual reproduction. This approach identified 532 genes that contribute to sex, including more than 200 that were not previously annotated to be involved in the process, of which more than 150 have orthologs in vertebrates. Among our verified hits was an uncharacterized gene, ifs1 (important for sex), that is required for spore viability. In two other hits, plb1 and alg9, we observed a novel mutant phenotype of poor spore health wherein viable spores are produced, but the spores exhibit low fitness and are rapidly outcompeted by wildtype. Finally, we fortuitously discovered that a gene previously thought to be essential, sdg1 (social distancing gene), is instead required for growth at low cell densities. Our assay will be valuable in further studies of sexual reproduction in S. pombe and identifies multiple candidate genes that could contribute to sexual reproduction in other eukaryotes, including humans.

INFLUENCE OF CHITOSAN SOLUTION ON THE VIABILITY OF ARBUSCULAR MYCORRHIZAL SPORES

The study was aimed to fortify the Arbuscular Mycorrhizal Fungal (AMF) spores, widely used in Bio-fertilizers with chitosan and check the inuence of chitosan on the viability of AMF spores. Chitosan was prepared from shrimp shells using chemical method involving demineralization, deproteinization and deacetylation.AMF spores were kept in three different concentrations of chitosan (0.1%, 0.5 %and 1%) which was prepared in 0.1% acetic acid and 0.1% ascorbic acid. Spore viability was checked by the MTT 3-(4, 5 th th th dimethylthiazol-yl-2, 5-diphenyl-2H-tetrazolium bromide) after a day, 10 day, 20 day and 30 day. Highest number of viable spores was observed in chitosan dissolved in 0.1% ascorbic acid as compared to chitosan in acetic acid.

Pengendalian Hama Kelapa Larva Kumbang Badak (Oryctes rhinoceros, L.) Instar III Dengan Metarhizium anisopliae, Metch. yang Ditumbuhkan Pada Berbagai Macam Dedak Gandum

The research objectives were to observe the growth and spore production of Metarhizium anisopliae grown at wheat-pollard and wheat-brand and to test the M. anisopliae pathogenicity on instar III rhinoceros beetle larva of coconut pest (Oryctes rhinoceros, L.). The research were executed in 3 phases : (1) Regeneration phase of M. anisopliae, the experiment was arranged in single factor CRD with 3 kinds of wheat and 3 replications. The influences of the treatments on biomass, spore number and spore viability were observed then; (2) lethal dosage (LD50) determination phase; and (3) phatogenicity test of M. anisopliae, the experiment was arranged in 3 x 3 factorial CRD, where the first factor was the kinds of wheat (i.e. Pollard, Brand and Pollard Brand) and the second factor was the dosage of biopesticide (i.e. 0,5 LD50 LD50 and 2 LD50). The phatogenicity testing was conducted to evaluate the effects on toxicity, mortality speed, and effication percentage. The result indicated that the wheat-brand was the better medium for M. Anisopliae than the pollard and the pollard-brand ones. The average spore number (1021,67 x 1014 spora/ml), biomass (95,94 g) and the viability (127,00 x 108 spora/ml) were significantly higher than the others. The lethal dosage obtained was 6,70g to gave the mortality percentage (73,33 %) and the effication percentage (73,33 %) significantly higher than the other treatments.

The Inactivation by Curcumin-Mediated Photosensitization of Botrytis cinerea Spores Isolated from Strawberry Fruits

Photosensitization is a novel environmentally friendly technology with promising applications in the food industry to extend food shelf life. In this study, the natural food dye curcumin, when combined with visible light (430 nm), was shown to be an effective photosensitizer against the common phytopathogenic fungi Botrytis cinerea (the cause of grey mould). Production of the associated phytotoxic metabolites botrydial and dihydrobotrydial was measured by our newly developed and validated HRAM UPLC-MS/MS method, and was also shown to be reduced by this treatment. With a light dose of 120 J/cm2, the reduction in spore viability was directly proportional to curcumin concentrations, and the overall concentration of both botrydial and dihydrobotrydial also decreased with increasing curcumin concentration above 200 µM. With curcumin concentrations above 600 µM, the percentage reduction in fungal spores was close to 100%. When the dye concentration was increased to 800 µM, the spores were completely inactive and neither botrydial nor dihydrobotrydial could be detected. These results suggest that curcumin-mediated photosensitization is a potentially effective method to control B. cinerea spoilage, and also to reduce the formation of these phytotoxic botryane secondary metabolites.

Plasmodiophora brassicae in its environment-effects of temperature and light on resting spore survival in soil.

Clubroot caused by Plasmodiophora brassicae is an important disease on cruciferous crops worldwide. Management of clubroot is challenging, largely due to the millions of resting spores produced within an infected root that can survive dormant in the soil for many years. This study was conducted to investigate some of the environmental conditions that may affect the survival of resting spores in the soil. Soil samples containing clubroot resting spores (1 × 107 spores/g soil) were stored at various temperatures for two years. Additionally, other samples were buried in soil, or kept on the soil surface in the field. The content of P. brassicae DNA and the numbers of viable spores in the samples were assessed by quantitative polymerase chain reaction (qPCR) and pathogenicity bioassays, respectively. The results indicated that 4°C, 20°C, and being buried in the soil were more conductive conditions for spore survival compared to -20°C, 30°C, and at the soil surface. 99.99% of the spores kept on the soil surface were non-viable, suggesting a negative effect of light on spore viability. Additional experiments confirmed the negative effect of UV light on spore viability as spores receiving 2- and 3-hour UV light exhibited lower disease potential and contained less DNA content compared to the untreated control. Finally, this work confirmed that DNA-based quantification methods such as qPCR can be poor predictors of P. brassicae disease potential due to the presence and persistence of DNA from dead spores.

Nickel excess affects phenology and reproductive attributes of Asterella wallichiana and Plagiochasma appendiculatum growing in natural habitats

Bryophytes are potent metal absorbers, thriving well on heavy metal (HM)-polluted soils. Mechanisms controlling uptake, compartmentalization and impacts of HMs on bryophytes life cycle are largely unknown. The current study is an effort to decipher mechanisms of nickel (Ni) excess-induced effects on the phenological events of two bryophytes, Asterella wallichiana and Plagiochasma apendiculatum growing in natural habitats. Observations revealed Ni-excess induced negative impacts on abundance, frequency of occurrence of reproductive organs, population viability and morphological traits, spore viability and physiological attributes of both the liverworts. Results led us conclude that P. appendiculatum survived better with the lowest impact on its life cycle events than A. wallichiana under Ni excess in natural habitats. Our findings collectively provide insights into the previously unknown mechanisms of Ni-induced responses in liverworts with respect to phenological attributes, as well as demonstrate the potential of P. appendiculatum to survive better in Ni excess habitats.

HbxB Is a Key Regulator for Stress Response and β-Glucan Biogenesis in Aspergillus nidulans

Homeobox transcription factors are conserved in eukaryotes and act as multi-functional transcription factors in filamentous fungi. Previously, it was demonstrated that HbxB governs fungal development and spore viability in Aspergillus nidulans. Here, the role of HbxB in A. nidulans was further characterized. RNA-sequencing revealed that HbxB affects the transcriptomic levels of genes associated with trehalose biosynthesis and response to thermal, oxidative, and radiation stresses in asexual spores called conidia. A phenotypic analysis found that hbxB deletion mutant conidia were more sensitive to ultraviolet stress. The loss of hbxB increased the mRNA expression of genes associated with β-glucan degradation and decreased the amount of β-glucan in conidia. In addition, hbxB deletion affected the expression of the sterigmatocystin gene cluster and the amount of sterigmatocystin. Overall, these results indicated that HbxB is a key transcription factor regulating trehalose biosynthesis, stress tolerance, β-glucan degradation, and sterigmatocystin production in A.nidulans conidia.