Metarhizium robertsii protease and conidia production, response to heat stress and virulence against Aedes aegypti larvae

Nutritional factors exert significant influence on the growth of entomopathogenic fungi, one of the main agents employed commercially in the biological control of arthropods. Thus, the objective of this work is to optimize the culture medium and solid fermentation time for production of proteases and conidia of Metarhizium robertsii ARSEF 2575 and to evaluate the interference of riboflavin and salts on virulence and resistance to abiotic stress factors. In the first step, nine groups were separated: negative control, positive control, and seven supplementation groups: ammonium nitrate, ammonium chloride, potassium nitrate, sodium nitrate, ammonium sulfate, ammonium phosphate, urea. Sodium nitrate showed significant difference in protease production at the time of 20 days of solid fermentation. Then, different concentrations of sodium nitrate and riboflavin as supplement were evaluated. Response surface methodology demonstrated that riboflavin and sodium nitrate influence proteolytic activity and conidia production, but without synergism. Supplementation of the medium with the optimal concentration of sodium nitrate and riboflavin did not interfere with the germination of conidia without exposure to abiotic stress, but did increase the thermotolerance of conidia. The presence of riboflavin and sodium nitrate at optimal concentrations in the culture medium did not alter fungal virulence with and without exposure to heat stress, varying according to the presence or absence of the supernatant during exposure, evidencing that resistance to heat exposure is multifactorial and dependent on intra- and extracellular factors. Moreover, the supplementation increased the larvicidal activity of the supernatant against Aedes aegypti.

High Predatory Capacity of a Novel Arthrobotrys oligospora Variety on the Ovine Gastrointestinal Nematode Haemonchus contortus (Rhabditomorpha: Trichostrongylidae)

With the worldwide development of anthelmintic resistance, new alternative approaches for controlling gastrointestinal nematodes in sheep are urgently required. In this work, we identified and characterized native nematode-trapping fungi. We collected seven isolates of fungi with the capacity to form adhesive, three-dimensional networks as the main mechanism to capture, kill, and consume nematodes. The nematode-trapping fungi were classified into two groups; the first group includes the R2-13 strain, showing faster growth, abundant aerial hyphae, scarce conidia production, bigger conidia, and it formed a clade with Arthrobotrys oligospora sensu stricto. The second comprises the A6, A12, A13, R2-1, R2-6, and R2-14 strains, showing a growth adhering to the culture medium, forming little aerial hyphae, smaller conidia, and these formed a sister clade to A. oligospora. Except for the R2-6 strain, conidia production was induced by light. In all the strains, the predatory capacity against the sheep gastrointestinal nematode Haemonchus contortus was greater than 58% compared with the control group. The A6 and A13 strains were the most active against the infective H. contortus third instar (L3) larvae, with an average capture capacity of 91%. Altogether, our results support evidence for a novel A. oligospora variety with high nematode-trapping activity and promissory in helminthic control.

A Novel Partitivirus That Confer Hypovirulence to the Plant Pathogenic Fungus Colletotrichum liriopes

Here, we report a novel double-stranded RNA virus designated Colletotrichum liriopes partitivirus 1 (ClPV1) from the plant pathogenic fungus C. liriopes. ClPV1 genome has two double stranded RNAs (dsRNAs), named as dsRNA 1 and dsRNA 2, which in the lengths of 1,807 and 1,706 bp, respectively. The dsRNA 1 and dsRNA 2 encoded proteins showing significant amino acid (aa) sequence identity to the RNA-dependent RNA polymerase (RdRp) and coat protein (CP) of partitiviruses, respectively. Phylogenetic analysis using the aa sequences of RdRp and CP indicated that ClPV1 was grouped to members of the putative Epsilonpartitivirus genus in the Partitiviridae family. Spherical viral particles in approximately 35 nm in diameter and packaging the ClPV1 genome were isolated. Virus elimination and virus transfection with purified viral particles, and biological comparison revealed that ClPV1 could reduce the virulence and conidia production of C. liriopes. To the best of our knowledge, this is the first report of mycovirus in C. liriopes fungus.

The Regulatory Mechanism of Water Activities on Aflatoxins Biosynthesis and Conidia Development, and Transcription Factor AtfB Is Involved in This Regulation

Peanuts are frequently infected by Aspergillus strains and then contaminated by aflatoxins (AF), which brings out economic losses and health risks. AF production is affected by diverse environmental factors, especially water activity (aw). In this study, A. flavus was inoculated into peanuts with different aw (0.90, 0.95, and 0.99). Both AFB1 yield and conidia production showed the highest level in aw 0.90 treatment. Transcriptional level analyses indicated that AF biosynthesis genes, especially the middle- and later-stage genes, were significantly up-regulated in aw 0.90 than aw 0.95 and 0.99. AtfB could be the pivotal regulator response to aw variations, and could further regulate downstream genes, especially AF biosynthesis genes. The expressions of conidia genes and relevant regulators were also more up-regulated at aw 0.90 than aw 0.95 and 0.99, suggesting that the relative lower aw could increase A. flavus conidia development. Furthermore, transcription factors involved in sexual development and nitrogen metabolism were also modulated by different aw. This research partly clarified the regulatory mechanism of aw on AF biosynthesis and A. flavus development and it would supply some advice for AF prevention in food storage.

Identification of Putative Virulence Genes by DNA Methylation Studies in the Cereal Pathogen Fusarium graminearum

DNA methylation mediates organisms’ adaptations to environmental changes in a wide range of species. We investigated if a such a strategy is also adopted by Fusarium graminearum in regulating virulence toward its natural hosts. A virulent strain of this fungus was consecutively sub-cultured for 50 times (once a week) on potato dextrose agar. To assess the effect of subculturing on virulence, wheat seedlings and heads (cv. A416) were inoculated with subcultures (SC) 1, 23, and 50. SC50 was also used to re-infect (three times) wheat heads (SC50×3) to restore virulence. In vitro conidia production, colonies growth and secondary metabolites production were also determined for SC1, SC23, SC50, and SC50×3. Seedling stem base and head assays revealed a virulence decline of all subcultures, whereas virulence was restored in SC50×3. The same trend was observed in conidia production. The DNA isolated from SC50 and SC50×3 was subject to a methylation content-sensitive enzyme and double-digest, restriction-site-associated DNA technique (ddRAD-MCSeEd). DNA methylation analysis indicated 1024 genes, whose methylation levels changed in response to the inoculation on a healthy host after subculturing. Several of these genes are already known to be involved in virulence by functional analysis. These results demonstrate that the physiological shifts following sub-culturing have an impact on genomic DNA methylation levels and suggest that the ddRAD-MCSeEd approach can be an important tool for detecting genes potentially related to fungal virulence.

PENGARUH KOMPOSISI MEDIA TERHADAP PERTUMBUHAN JAMUR Nomuraea rileyi (FARLOW) SAMSON DAN PATOGENISITASNYA PADA Helicoverpa armigera HUBNER DAN Spodoptera litura F.

<p>ABSTRAK</p><p>Nomuraea rileyi adalah salah satu jamur entomopatogen yangpotensial mengendalikan hama Helicoverpa armigera dan Spodopteralitura pada tanaman kapas, tembakau, dan jarak kepyar. Di lapanganpernah ditemukan larva hama H. armigera dan S. litura yang terinfeksisecara alami oleh N. rileyi yang mengindikasikan bahwa N. rileyiberpotensi sebagai agens hayati. Sebelum N. rileyi dikembangkan sebagaiagens hayati, maka perlu diketahui metode perbanyakannya pada mediabuatan. Penelitian bertujuan untuk mengetahui komposisi media tumbuhyang sesuai untuk perbanyakan N. rileyi dan pengujian patogenisitasnyaterhadap H. armigera dan S. litura. Penelitian dilakukan di LaboratoriumFitopatologi dan Laboratorium Patogen Serangga Balai PenelitianTanaman Tembakau dan Serat Malang mulai bulan Mei sampai denganNovember 2009. Penelitian ini terdiri atas 2 pengujian, yaitu pengujiankarakter biologi N. rileyi dan patogenisitas pada ulat H. armigera dan S.litura. Dalam pengujian karakter biologi jamur diuji 4 macam mediaperbanyakan, yaitu: (1) Sabouraud maltose agar + ekstrak yeast (SMAY),(2) Sabouraud maltose agar + ekstrak yeast + ekstrak beras (SMAYB), (3)Sabouraud maltose agar + ekstrak yeast + ekstrak kentang (SMAYK), dan(4) Media lengkap untuk N. rileyi (MLNr), serta 2 tingkat suhu inkubasi,yaitu 23±1 dan 27±1ºC. Perlakuan disusun dalam rancangan acak lengkap(RAL) dengan lima kali ulangan. Setiap media disiapkan di dalam 10cawan petri per perlakuan dan masing-masing diinokulasi dengan 10 5konidia/ml. Parameter yang diamati adalah laju pertumbuhan jamur danproduksi konidia. Sedangkan dalam pengujian patogenisitas konidia N.rileyi terhadap larva H. armigera dan S. litura dilakukan dengan metodepelumuran (painting), yaitu ulat diletakkan di atas konidia di dalam cawanpetri selama ± 10 detik kemudian dipindahkan ke vial-vial plastikberdiameter 2,5 cm berisi pakan daun kapas muda (± 1 cm 2 ) untuk H.armigera dan daun jarak kepyar untuk S. litura. Apabila pakan daun telahhabis, serangga diberi pakan buatan berbahan dasar tepung kedelai. Pakanbuatan diganti setiap 2 hari sampai ulat menjadi pupa. Selanjutnya ulatyang telah diperlakukan dengan jamur diinkubasi-kan pada suhu ruang(27°-29°C) selama ± 14 hari dan diamati perkembangan ulat maupunjamurnya setiap hari. Parameter yang diamati adalah mortalitas ulat H.armigera dan S. litura serta gejala mikosis pada ulat terinfeksi. Hasilpenelitian menunjukkan bahwa suhu inkubasi berpengaruh terhadap lajupertumbuhan N. rileyi. Pada suhu 23±1ºC N. rileyi tumbuh lebih cepat(7,42-8,23 mm/hari) pada semua komposisi media yang diuji (SMAY,SMAYK, SMAYB, dan MLNr) dibanding pada suhu 27±1ºC (0,99-1,26mm/hari). Produksi konidia N. rileyi lebih banyak pada suhu 27±1ºCdibanding pada 23 ± 1ºC, yaitu berturut-turut 24,7 x 10 8 konidia/ml dan17,9 x 10 8 konidia/ml masing-masing pada media SMAYK dan MLNr.Perbedaan komposisi media tumbuh tidak menyebabkan penurunanpatogenisitas pada konidia N. rileyi sebab mortalitas ulat H. armigeramaupun S. litura masing-masing mencapai 100%. Hasil penelitianmengindikasikan bahwa N. rileyi mudah diperbanyak secara massal padamedium agar dan virulensinya baik pada H. armigera dan S. litura.</p><p>Kata kunci : Nomuraea rileyi, epizootik, Helicoverpa armigera,Spodoptera litura, konidia, patogenisitas, mortalitas</p><p>ABSTRACT</p><p>Effect of medium composition on growth of entomo-pathogenic fungi Nomuraea rileyi (Farlow) Samson andits pathogenicity against Helicoverpa armigera andSpodoptera litura</p><p>N. rileyi is one of potential entomopathogenic fungi to controlcotton bollworm, Helicoverpa armigera, tobacco and Rhicinus caterpilar,Spodoptera litura. These fungi naturally infect those insect pests indicatingtheir potential to be used as natural control agent. Techniques of in vitroproduction of these fungi need to be developed to find out their potentialagainst the insect target. Study on effect of medium composition ongrowth of entomopathogenic fungi N. rileyi and its pathogenicity againstH. armigera and S. litura was carried out at Phytopathology and InsectPathology Laboratories of Indonesian Tobacco and Fiber Crops ResearchInstitute (IToFCRI) from May to November 2009. The objective of thestudy was to find out the suitable composition of medium for N. rileyi andits pathogenicity against H. armigera and S. litura. The study consisted oftwo tests. The first test was testing for biological characters, namely invitro growth rate and conidia production of N. rileyi on four differentcompositions of medium as followed: (1) Sabouraud maltose agar + yeastextract (SMAY), (2) Sabouraud maltose agar + yeast extract + rice extract(SMAYR), (3) Sabouraud maltose agar + yeast extract + potato extract(SMAYP), and (4) Completed medium for N. rileyi (MLNr). Alltreatments were designed in randomized complete design (RCD) with fivereplicates. Parameters observed were the growth rate of N. rileyi andconidia production. The second was testing on pathogenicity of N. rileyiproduced from all medium tested against H. armigera and S. litura larvae.Result showed that incubation temperature influenced the growth rate offungi. N. rileyi grew faster at 23±1ºC (7.42-8.23 mm/day) than that at27±1ºC (0.99-1.26 mm/day) on all media tested. Conidia production washigher at 27±1ºC than at 23±1ºC. Both SMAYP and MLNr were the bestmedia for producing N. rileyi conidia, which were 24.7 and 17.9 x 10 8conidia/ml, respectively. Pathogenicity of N. rileyi against H. armigeraand S. litura was not affected by composition of medium tested becausethe larval mortality of both insect pests was 100%. This study indicatedthat N. rileyi can be easily produced massively on agar media and it isvirulent against H. armigera and S. litura.</p><p>Key words : Nomuraea rileyi, Helicoverpa armigera, Spodoptera litura,conidia, in vitro, pathogenicity, mortality, epizootic</p>