Identifikasi Isolat Khamir Berpotensi sebagai Agens Antagonis dan Uji Produksi Toksin Hemolisin

Identifikasi khamir dapat dilakukan secara konvensional maupun molekuler. Identifikasi secara konvensional membutuhkan waktu yang lama dan interpretasi hasilnya seringkali bersifat subyektif. Sementara identifikasi khamir dengan metode molekuler dapat memberikan hasil yang lebih akurat dan cepat. Khamir yang berperan sebagai agens antagonis harus aman terhadap organisme nontarget agar dapat diaplikasikan di lapangan. Penelitian ini bertujuan untuk mengidentifikasi isolat-isolat khamir berpotensi antagonis dengan metode molekuler dan mengetahui kemampuan khamir dalam menghasilkan hemolisin sebagai salah satu indikator potensi resiko terhadap mamalia. Identifikasi dan pengujian kemampuan khamir dalam menghasilkan hemolisin dilakukan pada 15 isolat khamir berpotensi antagonis terhadap patogen antraknosa cabai (Colletotrichum acutatum). Identifikasi khamir dilakukan secara molekuler dengan PCR menggunakan primer ITS1 dan ITS4. Penyediaan khamir menggunakan mediaYeast Malt Extract Broth (YMB) dan Potato Dextrose Agar (PDA). Pengujian kemampuan khamir dalam menghasilkan hemolisin menggunakan media blood agar base (Oxoid CM55) ditambah darah domba 5%. Hasil identifikasi menunjukkan bahwa isolat khamir dapat teramplifikasi dengan primer ITS1 dan ITS4 dengan ukuran fragmen produk antara 500-800 pb. Hasil analisis sekuensing didapatkan 6 spesies khamir yaitu Candida tropicalis, Rhodotorula minuta, Aureobasidium pullulans, Pseudozyma hubeiensis, Pseudozyma aphidis, dan Pseudozyma shanxiensis. Uji kemampuan khamir dalam menghasilkan hemolisin menunjukkan bahwa seluruh khamir yang diuji tidak menghasilkan toksin hemolisin sehingga diduga isolat-isolat tersebut tidak patogenik terhadap manusia.

Secretion-Based Modes of Action of Biocontrol Agents with a Focus on Pseudozyma aphidis

Plant pathogens challenge our efforts to maximize crop production due to their ability to rapidly develop resistance to pesticides. Fungal biocontrol agents have become an important alternative to chemical fungicides, due to environmental concerns related to the latter. Here we review the complex modes of action of biocontrol agents in general and epiphytic yeasts belonging to the genus Pseudozyma specifically and P. aphidis in particular. Biocontrol agents act through multiple direct and indirect mechanisms, which are mainly based on their secretions. We discuss the direct modes of action, such as antibiosis, reactive oxygen species-producing, and cell wall-degrading enzyme secretions which can also play a role in mycoparasitism. In addition, we discuss indirect modes of action, such as hyperbiotrophy, induced resistance and growth promotion based on the secretion of effectors and elicitors from the biocontrol agent. Due to their unique characteristics, epiphytic yeasts hold great potential for use as biocontrol agents, which may be more environmentally friendly than conventional pesticides and provide a way to reduce our dependency on fungicides based on increasingly expensive fossil fuels. No less important, the complex mode of action of Pseudozyma-based biocontrol agents can also reduce the frequency of resistance developed by pathogens to these agents.

Karakterisasi Morfologi dan Pemanfaatan Sumber Karbon oleh Khamir Antagonis Patogen Antraknosa

Karakterisasi morfologi khamir penting dilakukan sebagai pengenalan awal suatu isolat khamir. Sedangkan, pengamatan terhadap pemanfaatan sumber karbon digunakan untuk mengetahui sumber karbon yang dapat digunakankan oleh khamir untuk perbanyakan dan peningkatan kemampuannya sebagai agens biokontrol. Penelitian ini bertujuan untuk mengkarakterisasi morfologi dan mengetahui pemanfaatan sumber karbon oleh khamir antagonis patogen antraknosa pada cabai (Colletotrichum acutatum). Karakterisasi morfologi khamir dilakukan dengan mengamati secara makroskopis koloni enam spesies khamir dalam medium PDA. Karakterisasi morfologi juga dilakukan dengan mengamati mikroskopis sel khamir di bawah mikroskop cahaya photomicrograph multi eyepiece (Zeiss Axiocam). Pengamatan pemanfaatan sumber karbon oleh khamir dilakukan dengan menggunakan BIOLOGTM (MicrologTM System, Release 5.2). Karakteristik morfologi enam spesies khamir antagonis menunjukkan bahwa koloni khamir didominasi warna krem keputihan dengan permukaan kasar, kusam, dan tepi koloni tidak rata. Karakteristik sel secara mikroskopis menunjukkan bentuk sel didominasi oleh bentuk silinder, dengan ukuran bervariasi. Sebagian besar khamir bersifat dimorfik. Pengamatan pemanfaatan sumber karbon oleh khamir antagonis menunjukkan Aureobasidium pullulans, Pseudozyma hubeiensis, Pseudozyma aphidis dan Pseudozyma shanxiensis dapat memanfaatkan 20 sumber karbon yang sama yaitu Tween 80, Arbutin, D-Gluconic Acid, D-Glucuronic Acid, D-Ribose, Salicin, ɤ-AminoButyric Acid, Bromosuccinic Acid, Fumaric Acid, β-Hydroxy Butyric Acid, L-Lactic Acid, LMalic Acid, Succinamic Acid, Succinic Acid, Alaninamide, L-Alanine, L-Aspartic Acid, LGlutamic Acid, Putrecine, dan Quinic Acid. Sedangkan, dua spesies khamir yaitu Rhodotorula minuta dan Candida tropicalis dapat memanfaatkan dua sumber karbon yang sama yaitu D-Trehalose dan D-Galactose plus D-Xylose.

Molecular identification of Pseudozyma aphidis (Henninger & Windisch) Boekhout: first record from a Brazilian mangrove swamp

ABSTRACT (Molecular identification of Pseudozyma aphidis (Henninger & Windisch) Boekhout: first record from a Brazilian mangrove swamp). Pseudozyma aphidis (Henninger & Windisch) Boekhout is an anamorph yeast-like basidiomycete commonly found as saprotroph in vegetable debris, such as leaves and flowers. However, there are also reports that this species is pathogenic in humans, usually occurring in patients with some type of immunosuppression, which predisposes to opportunistic infections. Specimens of P. aphidis were collected from water samples and Rhizophora mangle L. leaves at different salinities along the Perequê River, located in a mangrove swamp area at Parque Estadual da Ilha do Cardoso, Cananéia municipality, São Paulo State, Brazil. Aliquots of these samples were spread in Petri dishes. The samples baited with cataphylls of Allium cepa L. were incubated for seven days at 21 ºC. After incubation, the baits were observed under microscope and the specimens were isolated and purified. The identification of the specimens was made through the phylogenetic analysis of the ITS rDNA region. This is the first record of P. aphidis in São Paulo State.

Foliar microbiome transplants confer disease resistance in a critically-endangered plant

There has been very little effort to incorporate foliar microbiomes into plant conservation efforts even though foliar endophytes are critically important to the fitness and function of hosts. Many critically endangered plants that have been extirpated from the wild are dependent on regular fungicidal applications in greenhouses that cannot be maintained for remote out-planted populations, which quickly perish. These fungicides negatively impact potentially beneficial fungal symbionts, which may reduce plant defenses to pathogens once fungicide treatments are stopped. Using the host/parasite system of Phyllostegia kaalaensis and Neoerysiphe galeopsidis, we conducted experiments to test total foliar microbiome transplants from healthy wild relatives onto fungicide-dependent endangered plants in an attempt to mitigate disease and reduce dependency on fungicides. Plants were treated with total microbiome transplants or cultured subsets of this community and monitored for disease severity. High-throughput DNA screening of fungal ITS1 rDNA was used to track the leaf-associated fungal communities and evaluate the effectiveness of transplantation methods. Individuals receiving traditionally isolated fungal treatments showed no improvement, but those receiving applications of a simple leaf slurry containing an uncultured fungal community showed significant disease reduction, to which we partially attribute an increase in the mycoparasitic Pseudozyma aphidis. These results were replicated in two independent experimental rounds. Treated plants have since been moved to a native habitat and, as of this writing, remain disease-free. Our results demonstrate the effectiveness of a simple low-tech method for transferring beneficial microbes from healthy wild plants to greenhouse-raised plants with reduced symbiotic microbiota. This technique was effective at reducing disease, and in conferring increased survival to an out-planted population of critically endangered plants. It was not effective in a closely related plant. Plant conservation efforts should strive to include foliar microbes as part of comprehensive management plans.