Partial Purification and Characterization of Chitinase from Entomopathogenic Fungus Beauveria bassiana Isolate BB200109. Yadi Suryadi, Tri P. Priyatno, I Made Samudra, Dwi N. Susilowati, Nuni Lawati, and Eman Kustaman. Beauveria bassiana is one of the entomopathogenic fungus that produces chitinase when infecting its host. This study was aimed to purify, isolate and characterize chitinase of B. bassiana isolate BB200109. Pathogen identity was determined both morphologically and molecularly using ITS primer, whilst characterization was done at various conditions i.e. temperature, pH, metal ion and incubation time. Results showed that the BB200109 isolate belonged to B. bassiana. The isolate produced extracellular chitinase with chitinolytic index of 1.035. Partial purification of three saturated ammonium sulphate precipitation (10, 30, and 70%) showed maximum purity of 1.2 times, while dialysis could increase the purity of 1.9 times compared to that of crude enzyme extract. Characterization results showed that the chitinase isolated from B. bassiana isolate BB200109 had an optimum activity at pH 4, temperature 50 o C, and optimum incubation time of 90 minutes. , and Na + acted as inhibitors. The chitinase demonstrated lower affinity to chitin substrate as indicated by high K m value of 0.266 mg/l and a V max of 0.067 mg/l sec. Based on SDS-PAGE, chitinase from B. bassiana isolate BB200109 had molecular weight of 60.25 kDa. The study implied the potency of B. bassiana isolate BB200109 as extracellular chitinase producer with its enzyme charateristics seems to be developed as an insect biocontrol agent.

The cyclic AMP- (cAMP-) dependent protein kinase A signaling pathway is one of the major signaling pathways responsible for regulation of the morphogenesis and pathogenesis of several pathogenic fungi. To evaluate the role of this pathway in the plant pathogenic fungus, Colletotrichum gloeosporioides, the gene encoding the catalytic subunit of cAMP-dependent protein kinase A, CgPKAC, was cloned, inactivated, and the mutant was analyzed. Analysis of the Cgpkac mutant generated via gene replacement showed that the mutants were able to form appressoria; however, their formation was delayed compared to the wild type. In addition, the mutant conidia underwent bipolar germination after appressoria formation, but no appressoria were generated from the second germ tube. The mutants also showed reduced ability to adhere to a hydrophobic surface and to degrade lipids localized in the appressoria. Based on the number of lesions produced during a pathogenicity test, the mutant's ability to cause disease in healthy mango fruits was reduced, which may be due to failure to penetrate into the fruit. These findings indicate that cAMP-dependent protein kinase A has an important role in regulating morphogenesis and is required for pathogenicity of C. gloeosporioides.

ABSTRACT

<p>Anthracnose (Colletotrichum gloeosporioides) is one of the important diseases of fruit crops that need to be controlled. This study was aimed to obtain the best formula of hydrolyzed nano chitosan and its potensial in controlling anthracnose. The hydrolyzed chitosan was prepared using chitinase enzyme extracted from Burkholderia cepacia isolate E76. Chitosan nanoparticles were synthesized using ionic gelation method by reacting hydrolyzed chitosan (0.2%) with Sodium tripolyphosphate (STPP) (0.1%) as cross-linking agent using 30&amp;ndash;60 minutes stirring condition. The bioactivity of the nano chitosan formula was tested to C. gloeosporioides under in vitro and in vivo assays. The specific enzymatic activity of the purified chitinase was higher (0.19 U/mg) than that of crude enzyme (supernatant) with the purity increased by 3.8 times. Of the four formula tested, Formula A (hydrolyzed chitosan to STPP volume ratio of 5 : 1 with 60 minutes stirring condition) was found good in terms of physical characteristic of the particle. The formula nano chitosan particle had the spherical-like shape with an average particle size of 126.2+3.8 nm, polydispersity index (PI) of 0.4+0.02, and zeta potential (ZP) value of 27.8+0.2 mV. Nano chitosan had an inhibitory activity to C. gloeosporioides in vitro up to 85.7%. Moreover, it could inhibit 61.2% of C. gloeosporioides spores germination. It was shown that nano chitosan was also effective to reduce anthracnose disease severity in vivo when applied as a preventive measure on chili and papaya fruits. This study could be used as a reference for further fruit coating application using nano chitosan as a promising postharvest biocontrol agent to C. gloeosporioides.</p>

No abstract