CC BY
50
Ukrainian Journal of Ecology
Ukrainian Journal of Ecology, 2017, 7(4), 689-691, doi: 10.15421/2017_1801
ORIGINAL ARTICLE
Pathogenicity of Simplicillium lanosoniveum to Coccus hesperidum
M. Skaptsov1, S. Smirnov1,2, M. Kutsev1, O. Uvarova1, T. Sinitsyna1, A. Shmakov1, A. Matsyura1
1 Altai State University, Barnaul, 656049, Russia, E-mail. [email protected] , m [email protected], [email protected] , [email protected] , ssbgbot@mail. r , amatsyura@gmai. com 2PlantaBio LLC, Barnaul, Russia, E-mail: serg sm @mail.ru Submitted: 06.11.2017. Accepted:24.12.2017
Simplicillium lanosoniveum isolate SSBG2 was isolated from the diseased C hesper collected from Schefflera octophylla in greenhouse of the South-Siberian Botanical Garden, and identified based on morphological observation and ITS region analysis. The infected plants were inoculated with conidial suspension of the isolate SSBG2 in concentrations 1.0*105/mL. It was showed that after inoculation the C hesperfemale adults were more vulnerable to infection. Larvae are affected to a lesser extent. The mycelium grows under the scale and cause the death of the insect. It was indicated that S. lanosoniveum had high infectivity against C hesper. Infection symptoms appeared on day 7 after the inoculation, the infection reached the peak on day 20. Our study provides a new isolate that affects the Coccus hesperidum. Key words: Coccus; entomopathogenic; fungi; PCR; Simplicillium
Introduction
The entomopathogenic fungi are parasitic microorganisms of various arthropods and causing acute mycoses. They can spread fast via aerial conidia and infect their host by penetration of the cuticle, grow within the internal fluids and produce toxins which kill the host. After that, the mycelium grows throughout to completing the life cycle by abundant conidial formation. Fungal species can infect a broad range of insect and several biopesticide products are developed for use in agriculture. The isolation and identification of native entomopathogenic fungi are necessary to provide a pool of biological agents for pest control. Fungal species from other areas may not be effective due to environmental differences (Lockwood, 1993). Beauveria, Metarhizium, Lecanicillium are among the most common bioagents. Simplicillium lanosoniveum is not widely used. Data on its interaction with other organisms are accumulating only now.
Materials and methods
S. lanosoniveumwas isolated from greenhouse of the South-Siberian Botanical Garden. The insects were transferred to potato-dextrose agar (PDA) and incubated at 25 °C, under diffuse light. The individual fungus colony were transferred to fresh media until a clear culture was isolated. Greenhouse experiment was carried out at 25 °C, 95 % humidity for 10-14 days. To the Petri dish was added 1 % solution of Tween 20, then colony with aerial conidia were scraped from the agar. Conidial suspension was diluted to concentration 105/mL, after that the infected plants were treated. Identification was carried out morphologically and by PCR analysis. For isolate, a portion of a colony was scraped from the agar plates and transferred into a 1.5-ml Eppendorf tube. DNA was isolated by DiamondDNA kit (ABT LLc., Russia). The primers ITS1 5'- TCCGTAGGTGAACCTGCGG -3', ITS4 5'-TCCTCCGCTTATTGATATGC -3' were used for amplification (White et al., 1990). PCR(s) were carried out in 25 mkl reaction mix which included 5 ng DNA, 2.5 mkl 10x PCR buffer and 25 mM MgCl2 (Sibenzyme Llc., Russia), 1 |jL 5mM of mix dNTPs (Medigen Llc., Russia), 1 mkl of each 10 mM primer and 1 unit Taq DNA polymerase (Sibenzyme Llc., Russia) in the MyCycler thermal cycler (Bio-Rad, USA) using protocol: 94.0 °C for 5 min. [94.0 °C for 30 sec., 56.0 °C for 30 sec., 72 °C for 1 min.]x35, 72.0 °C for 5 min., 4.0 °C until the end of the process. PCR products were purified using spin columns. Sequencing by Sanger was conducted in the Syntol LLc., Russia. The sequences of ITS (MG807436) obtained in this study have been deposited in GenBank. Similarity checks were done at NCBI website. For further analysis, sequences of LSU and ITS of closely related sequences were
Ukrainian Journal of Ecology
690
downloaded from NCBI. Alignment was done using Muscle, MEGA 7.0 (Edgar, 2004). Phylogenetic analysis was done using Neighbor-joining statistical method with 10000 Bootstrap replications (Saitou, Nei, 1987; Kumar et al., 2016).
Results and discussion
Phylogenetic analysis using ITS region recovered the fungus in a good-supported (86 %) clade with species of Simplicillium lanosoniveum(EF513003.1). The sequence of Beauveria bassiana (KC253900.1) was used as outgroup (Fig. 1).
86
34
42
Simplicillium isolate SSBG2
■ EF513003.1_Sirnplicillium lanosoniveum
AB603991.1 Simplicillium minatense
AB6Ü3993.1 Simplicillium minatense I— AB6Ü4005.1 Simplicillium cylindrosporurn
— AB758126.1 Simplicillium lanosoniveum AB604001.1 Simplicillium subtropicum
— JX232277.1 Simplicillium obclavatum JX500428.1 Lecanicillium fungicola AB604004.1 Simplicillium aogashirnaense -KC253900.1 Beauveria bassiana
100
0.0100
H
Fig. 1. Phylogenetic analysis based on ITS region of the Simplicillium isolate SSBG2 (MG807436).
The isolate showed a good growth rate on the PDA agar. Mature conidia were formed 3-4 days after inoculation. When C. hesperidum were inoculated in greenhouse with the prepared S. lanosoniveum spore suspension, the insects did not show signs of infection. Mycelia were observed under scale of the adult female C. hesperidum after 7 days of inoculation (Fig. 3). The active growth of the mycelium, the larvae damage and reduced the number of larvae were observed on 20 days after inoculation (Fig. 2).
Fig. 2. Simplicillium lanosoniveum. a. Agar plate; b. Light microscopic analysis of mycelia and conidia; c-d. Entomopathogenicity assay in Coccus hesperidum (greenhouse experiment); S. lanosoniveum mycelia under scale of C. hesperidum.
691
Pathogenicity of Simpliciilium lanosoniveum
Most studies have been devoted to its impact on rust fungi. S. lanosoniveum hyper parasitism was observed on Aecidium elaeagni-latifoliae isolated in India (Baiswar et al., 2014).
Colonization by the S. lanosoniveum was found on a rust fungus Phakopsora pachyrhizi (Ward, 2011). Simpiiciliium was associated with Hemileia vastatrix and Uromyces pencanus (Chen et al., 2017). In vitro tests showed virulence of the S. lanosoniveum to the silkworm. S. lanosoniveum activity is noted to be comparable with Beauveria bassiana (Lim et al., 2014). As our work, but on the other scale insects, entomopathogenicity was noted for Pseudaulacaspispentagona (Wang, 2016). S. lanosoniveum isolate Cs0701 had high virulence against the aphids, Aphisgossypii and Ceratovacuna lanigera (Chen et al., 2017). Some works reported that S. lanosoniveum can be pathogen of plant parasitic nematodes (Liu, Cai, 2012; Zhao et al., 2013). However, according to some sources, the S. lanosoniveum can be a cause of plant diseases. Symptoms of the disease included many irregular, dark brown spots on both upper and lower leaf surfaces were found on the Salvinia auricula ta and S. molesta in Taiwan. Of these, isolates of fungi were obtained and identified morphologically as S. lanosoniveum (Chen et al., 2008). In some works, a symbiotic role of S. lanosoniveumwas identified. For example, for the blue-green alga Chroococcussp. the phenomenon of mutualism with S. lanosoniveumwas noted. In this case, phototrophic Cyanobacterium provides carbon and energy source for S. lanosoniveum; as in the aquatic systems, carbon source is relatively deficient and usually the key factor limiting the growth of heterotrophic fungus (Dong et al., 2013). Therefore, a broad assessment of pathogenicity of S. lanosoniveum on other organisms needs to be further studied. Our work presents the first data on the pathogenicity of S. lanosoniveum in relation to C. hesperidum.
References
Baiswar, P.S., Ngachan, V., Rymbai, H., Chandra, S. (2014.) Simplicillium lanosoniveum, a hyperparasite on Aecidium elaeagni-iatifoiiae in India. Austral. PlantDis, Note, 9, 144. doi: 10.1007/s13314-014-0144-z
Chen, R., Huang, C., Li, J, Tsay, J. (2017). Evaluation of characteristics of Simpiiciliium lanosoniveum on pathogenicity to aphids and in vitro antifungal potency against plant pathogenic fungi. Int. J Environ. Agric. Res., 3(1), 2454-1850. Chen, R., Huang, C., Li, J., Tsay, J. (2008). First report of Simplicillium lanosoniveum causing brown spot on Salvinia auriculata and S. molesta in Taiwan. Plant disease, 92(11), 1589. doi: 10.1094/PDIS-92-11 -1589C
Dong, Q.L., Lin, T.Y., Xing, X.Y., Chen, B., Han, Y. (2013). Identification of a symbiotic fungus from blue-green alga and its extracellular polysaccharide. Lett App. Microbiol., 58, 303-310. doi: 10.1111 /lam.12192
Edgar, R.C. (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput. NucleicAcids Res., 32(5), 1792-1797. doi: 10.1093/nar/gkh340
Kumar, S., Stecher, G., Tamura, K. (2016). MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets. Mol. Biol. Evol., 33, 1870-1874. doi: 10.1093/molbev/msw054
Lim, S.Y., Lee, S., Kong, H.G., Lee, J. (2014). Entomopathogenicity of Simpliclllium lanosoniveum isolated in Korea. Mycobiology, 42(4), 317-321. doi: 10.5941 /MYC0.2014.42.4.317
Liu, W., Xie, Y., Xue, J., Gao, Y., Zang, Y., Zhang, X., Tan, J. (2009). Histopathological changes of Ceroplastesjaponicusinfected by Lecaniciliium lecanii. J Invertebr. Pathol., 101, 96-105. doi: 10.1016/j.jip.2009.03.002
Lockwood, D.A. (1993). Environmental issues involved in biological control of rangeland grasshopper (Orthoptera: Acrididae) with exotic agents. Environ. Entomology 22, 503-518. doi: 10.1093/ee/22.3.503
Saitou, N., Nei, M. (1987). The neighbor-joining method: A new method for reconstructing phylogenetic trees. Mol. Biol. Evol., 4, 406-425. doi: 10.1093/oxfordjournals.molbev.a040454
Wang, N., Xie, Y., Fan, J. (2016). Pathogenicity of Simplicillium lanosoniveumTYL001 isolated from Pseudaulacaspispentagona. Mycosystema, 35(5), 559-568.
Ward, N.A., Schneider, R.W., Aime, M.C. Colonization of soybean rust sori by Simpiicillium lanosoniveum. FungalEcol., 4, 303308. doi: 10.1016/j.funeco.2011.03.008
White, T.J., Bruns, T., Lee, S., Taylor, J. (1990). Amplification and direct sequencing of fungi ribosomal RNA genes for phylogenetics. In M.A. Innis, D.H. Gelfand, J.J. Sninsky and T.J. White (Eds.), PCR Protocols. A Guide to Methods and Applications (pp. 315-322). San Diego: Academic Press.
Zhao, D., Liu, B., Li, L.Y., Zhu, X.F., Wang, Y.Y., Wang, J.Q., Duan, Y.X., Chen, L.J. (2013). Simpiiciliium chinense. a biological control agent against plant parasitic nematodes. BiocontroiSci. Techn, 23, 980-986. doi: 10.1080/09583157.2013.809514
Citation:
Skaptsov, M., Smirnov, S., Kutsev, M., Uvarova, O., Sinitsyna, T., Shmakov, A., Matsyura, A. (2017). Pathogenicity of Simplicillium lanosoniveum to Coccus hesperidum. Ukrainian Journal of Ecology, 7{4), 689-691. I ("OE^^MIThk work Is licensed under a Creative Commons Attribution 4.0. License
