Evaluation of anti-Fusarium and auxin production of Trichoderma virens InaCC F1030 isolated from rhizosphere of banana

Banana rhizosphere harbors a unique diversity of microbes including fungi that play critical roles in the growth of the plant host as well as might be important for biologically controlling the fungal soil-borne pathogens particularly Fusarium oxysporum f.sp. cubense (Foc), the causing agent of devastating Panama wilt. Among other fungi, we have succeeded to isolate a Trichoderma species from rhizosphere of healthy banana. Molecular identification revealed the isolate as Trichoderma virens InaCC F1030 (being collection of Indonesian Culture Collection or InaCC). Therefore, the aim of this study was to investigate the biological control of our isolate against Foc as well as plant growth promoting ability through its ability to produce auxin (indole-3-acetic acid/IAA). Two approaches were employed to evaluate the antagonism of our isolate against Foc, through direct confrontation test and volatile organic compounds (VOCs) producing. We found that our isolate was considered as antagonistic to the Foc, but not highly antagonistic according to direct confrontation assay. However, it was also revealed that our isolate produces the VOCs that inhibited around 50% of the mycelial growth of the test pathogen after six to seven days of exposure. Our isolate was able to produce the IAA in axenic submerged fermentation condition particularly in the presence of the precursor L-tryptophan. IAA production ability as well as the mycelial biomass of fungus were increased approximately 17% and 120% respectively as the effect of supplementation of 0.1% of L-tryptophan. These in vitro bioassays lead us to conclude that somehow our isolate T. virens InaCC F1030 has potency to be utilized as biocontrol and biofertilizer agent

The effect of substrate composition on the activity of amylase and cellulase by Trichoderma harzianum strains under solid state fermentation

Trichoderma harzianum is a filamentous fungus that has been known to have biocontrol and plant growth-promoting ability. However, the propagation of this fungus particularly through solid state fermentation (SSF) and characterization of its enzyme activity as one the indicator of quality of fermentation process are still needed to be explored further. Rice grain and its derivative products have economically as well as nutrient composition features beneficial as substrates of fungal propagation through SSF. Therefore, the aim of this study was to investigate the effect of white rice, rice bran, and combination of white rice and rice bran on the activity of amylase and cellulase by Trichoderma harzianum strains under SSF. Two strains of the fungus, InaCC F116 and InaCC F89, as well as their consortium were employed as fungal inoculants. After closed fermentation in dark chamber at 30 ± 1°C for 7 days, the activity of amylolytic as well as cellulolytic enzyme was assayed. The result showed that the presence of rice bran as a substrate increased the activity of crude amylase and crude cellulase. In all substrates, the strain F116 has low activity of both enzymes. The fungal consortium improves the activity of crude enzymes in all substrates. Therefore, the amylase and cellulase activity by T. harzianum in SSF condition were strain- as well as substrate-dependent

Mycelial Growth-promoting Potential of Extracellular Metabolites of Paraburkholderia spp. Isolated from Rhizopogon roseolus Sporocarp

This study aimed to investigate the effect of potential metabolite(s) produced by Paraburkholderia spp. isolated from the Rhizopogon roseolus (shouro mushroom) sporocarp on the mycelial growth of R. roseolus. For this purpose, we selected two molecularly identified bacteria: P. fungorum GIB024 and P. caledonica KN1. Direct confrontation assay at three different distances, a pour plate method that sampled bacterial spent broth either with and without agitation at 25 °C, and an indirect confrontation assay was carried out in order to assess the R. roseolus growth-promoting ability of Paraburkholderia spp. These assessments were carried out in a 1:5 diluted Melin-Norkran-modified medium with glucose (hs-dMMN) and without glucose (ls-dMMN). GIB024 promoted the growth of R. roseolus in ls-dMMN in short distance, whereas KN1 inhibited the growth of the fungus in that condition. In hs-dMMN, both bacteria have neutral or slightly promotion effect toward R. roseolus. We determined from the spent broth analysis that Paraburkholderia spp. that grew axenically under static conditions had a more pronounced mycelial growth-promoting effect on R. roseolus than under agitation conditions. We also found that high concentration of spent broth resulted in a decrease in mycelial growth-promoting ability. Volatile metabolite(s) produced by both bacteria did not promote the mycelial growth of R. roseolus. In conclusion, Paraburkholderia spp. exhibited a species- and nutrient (sugar)-dependent ability to promote the mycelial growth of R. roseolus, and the bacterial soluble metabolite(s) play a crucial role in their growth-promoting ability

Biocontrol Activity, Mode of Action, and Colonization of Aureobasidium pullulans Dmg 30 DEP on Controlling Early Blight Disease on Tomato Plant

We investigated the biocontrol activity of A. pullulans Dmg 30 DEP against Alternaria solani causal agent of early blight. Biocontrol activity was tested by the in vivo and ad planta. Biocontrol activity were tested by investigating the antibiosis capabilities with dual culture method, paper dish assay, two-compartment petri dish assay, and trapping and identification of volatile organic compounds (VOCs) with GC-MS. Lysis activity was examined by observing the clear zone formed by growing yeast on chitin agar and skim milk agar. The ability of hyperparasitism was assessed by the agar block method, and observed by light microscopy and scanning electron microscopy (SEM). The results showed that A. pullulans Dmg 30 DEP plays a role in the suppression of early blight disease at 106 cells/ml and 107 cells/ml yeast cell density. The mechanism involved in biocontrol activity is the production of VOCs, the production of chitinase and protease enzymes, the production of siderophore and hyperparasitism. The result shows that A. pullulans Dmg 30 DEP was colonizing the tomato leaves following the areole

Investigation of the Physiological Function of Protein Phosphatase 4 (PP4) in Arabidopsis thaliana

Master's thesis in Biological chemistryProtein Phosphatase 4 (PP4), a member of serine/threonine-specific phospho-protein phosphatases (PPP) family, is remarkably well conserved across eukaryotes. PP4 has been studied mainly in yeast and mammalian cells, and virtually nothing is known about PP4 in plants. In mammalian cells, PP4 plays a role in several processes not relevant to plants. The major objective of this thesis was to investigate the physiological function of PP4 in Arabidopsis thaliana as a model plant. A. thaliana has two PP4 catalytic subunits, namely PP4-1 (At4G26720) and PP4-2 (At5G55260). In addition, putative regulatory subunits for PP4 were bioinformatically detected in A. thaliana: PP4R2L (At5G17070) and PSY2L (At3G06670). Using reverse genetics approach, this thesis focuses on expression studies (semiquantitative RT-PCR) and observation of phenotype of the A. thaliana gene encoding PP4 catalytic and putative regulatory subunits in loss-of-function mutants (T-DNA insertional mutagenesis lines), artificial microRNA (amiRNA) stable lines, and gene overexpression lines. Using intensive expression analysis by the in-gel RT-PCR, we succeeded to detect knock down and/or knock out for amiRNA plants and T-DNA plants for PSY2L and PP4R2L. Moreover, we detected and isolated stable overexpression plants for PP4-1, PP4-2, and PP4R2L. Observation of phenotype showed that knock out of PSY2L gene in a T-DNA line, SALK_048064 (insertion in exon 3 of 25), show some interesting phenotypes. The homozygous mutants of this line showed dwarfism, delayed growth, and extended life span. The knock down of PSY2L through amiRNA mechanism also showed a phenotype, such as reduced size and twisted leaves. No significant phenotype was found in overexpressor plants. We also investigated the subcellular localization of these subunits in two different plant expression systems: A. thaliana mesophyll protoplasts and particle bombardment into onion epidermis cells. Main location of the catalytic subunits, PP4-1 and PP4-2, are in cytoplasm, with few in nucleus. PSY2L is strongly found in nucleus, whereas the other regulatory subunit, PP4R2L is not only found in nucleus but also tend to locate in cytoplasm. In vivo investigations of subcellular localization of PP4 subunits show resemblance to in silico analysis

The influence of biocarrier of Aspergillus niger and Trichoderma harzianum toward vegetative growth of sorghum in the field experiment

Compared to other cereal crops, sorghum has a higher drought tolerance trait. However, efforts are needed to increase the productivity of sorghum, particularly in drought marginal land. One strategy to be implemented is the utilization of soil microorganisms formulated with biocarrier. Therefore, the aim of this study was to evaluate the influence of the fungal strain Aspergillus niger and Trichoderma harzianum formulated with compost and zeolite as biocarrier towards vegetative growth of sorghum. The field experiment was designed as a randomized block designed, factorial pattern with 4 replications. The first factor was selecting biocarrier, namely zeolite, compost, and a mixture of zeolite: compost (1:1). The second factor was the fungal inoculants, A. niger, and T. harzianum. The observed parameter was the growth profile of sorghum during vegetative growth, including stalk diameter and height. The results showed that the type of biocarrier, as well as the fungal strains did influence the growth of sorghum. The highest stalk diameter and height of sorghum were obtained after application of A. niger formulated with a mixture of zeolite: compost (1:1), with 17% and 41.2% higher than control, respectively. This condition shows that a mixture of zeolite and compost is seemingly able to create better micro-ecological conditions for fungal microbes to function effectively. Therefore, our findings suggested the addition of zeolite to compost for the application of biocarrier in the field experiment

THE PHSYIOLOGICAL CHARACTER OF BACTERIA ISOLATED FROM BANANA’S RHIZOSPHERE FROM MALAKA, EAST NUSA TENGGARA, AND THEIR ROLE ON PLANT GROWTH PROMOTION ON MARGINAL LAND

The objective of the study was to isolate microorganism that can produce growth hormone, fix atmospheric nitrogen, and solubilize inorganic phosphate that may be used for enhancing the growth of banana in marginal land.  A total of 25 bacteria associated with banana that belonged to six genera were isolated from namely Acinetobacter, Enterobacter, Pantoea, Klebsiella, Rhizobium, and Cupriavidus genera. Gammaproteobacteria is the dominant class, followed by Betaproteobacteria and Alphaproteobacteria. All isolates were screened for multiple plant growth promoting traits which may play a role in banana growth, namely fixing nitrogen, solubilizing phosphate, and producing Indole Acetic Acid (IAA). Twenty-two isolates were capable to fix nitrogen, 21 isolates can solubilize insoluble phosphate, and 15 isolates produced IAA dependent of L-Tryptophan presence. Despite most of the bacteria isolates exhibited one plant growth-promoting activities, Enterobacter and Klebsiella genera showed three of plant growth promoting bacteria traits.

Mycorrhiza stimulates Rhizobium infection in Paraserianthes falcataria (L.) I.C. Nielson under Hg contamination

Symbiosis of and AMF increase soybean production, but the information on the association between these microbes in Paraserianthes falcataria (L.) I.C. Nielson or ‘sengon’ under Hg contamination is limited. We observed P. falcataria inoculated with arbuscular mycorrhiza fungi (Glomus sp.) stimulate nodule formation under Hg contamination. The study was set up in a pot experiment in the growth chamber a the Hg concentration was adjusted to 50 ppm in soil medium. Inoculation of AMF stimulates nodule formation and increases P. falcataria tolerance to Hg. We isolated several bacteria from the rhizosphere belonging to Rhizobium group and others. Understanding the ecology of soil bacteria is important for Hg bioremediation using P. falcataria