A peer-reviewed version of this preprint was published in PeerJ on 6 October 2017.
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Meng X, Hu J, Ouyang G. 2017. The isolation and identification of pathogenic fungi from Tessaratoma papillosa Drury (Hemiptera: Tessaratomidae) PeerJ 5:e3888 https://doi.org/10.7717/peerj.3888 The isolation and identification of pathogenic fungus from the diseased Tessaratomapapillosa Drury (Hemiptera: Tessaratomidae) and their pathogenicity
Xiang Meng Corresp., 1 , Junjie Hu 2 , Gecheng Ouyang 1
1 Guangdong Institute of Applied Biological Resources, Guangzhou, Guangdong, China 2 College of Life Science, Guangzhou University, Guangzhou, Guangdong, China
Corresponding Author: Xiang Meng Email address: [email protected]
Background. Litchi stink-bug, Tessaratoma papillosa Drury (Hemiptera: Tessaratomidae) is a major pest on litchi and longan in Southern China. It is urgent to develop valid biological agent for control the pest and improve IPM strategy on orchard farming. Entomopathogenic fungi was regarded as avital ecological factor pressing pest populations in field, however, there were fewer searches conducted on entomopathogenic fungi against litchi stink-bug. Methods & Results. In this study, two pathogenic fungus were isolated from the adult diseased T. papillosa by normal methods and rDNA-ITS homogeneous analysis, they are identified as Paecilomyces lilacinus and Beauveria bassiana. Laboratory tests showed that the two entomopathogenic fungi both had a good lethal effect to young nymph and old nymph of T. papillosa. The toxicity determination showed that the LC50 value for Beauveria bassiana was higher than P. lilacinus. Conclusion.These pathogenic fungus did not have the risk of pollution or residue, and they can be a alternative option for integrated pest management approache.
PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.2851v1 | CC BY 4.0 Open Access | rec: 4 Mar 2017, publ: 4 Mar 2017 1 The isolation and identification of pathogenic fungus from the diseased Tessaratoma
2 papillosa Drury (Hemiptera: Tessaratomidae) and their pathogenicity
3
4 Xiang Meng 1, Junjie Hu 2 and Gecheng Ouyang 1
5
6
7 1 Guangdong Key Laboratory of Animal Conservation and Resource Utilization, Guangdong
8 Public Laboratory of Wild Animal Conservation and Utilization, Guangdong Institute of Applied
9 Biological Resources, Guangzhou, Guangdong Province, China.
10 2 College of Life Science, Guangzhou University, Guangzhou, Guangdong Province, China.
11
12 Correspondence
13 Gecheng Ouyang 1
14 Email addresses: [email protected]; [email protected]
15
16
17
18
19
20
21 22
23 Abstract：
24 Background. Litchi stink-bug, Tessaratoma papillosa Drury (Hemiptera: Tessaratomidae) is a
25 major pest on litchi and longan in Southern China. It is urgent to develop valid biological agent
26 for control the pest and improve IPM strategy on orchard farming. Entomopathogenic fungi was
27 regarded as avital ecological factor pressing pest populations in field, however, there were fewer
28 searches conducted on entomopathogenic fungi against litchi stink-bug.
29 Methods & Results. In this study, Two pathogenic fungus were isolated from the adult diseased
30 T. papillosa by normal methods and rDNA-ITS homogeneous analysis, they are identified as
31 Paecilomyces lilacinus and Beauveria bassiana. Laboratory tests showed that the two
32 entomopathogenic fungi both had a good lethal effect to young nymph and old nymph of T.
33 papillosa. The toxicity determination showed that the LC50 value for Beauveria bassiana was
34 higher than P. lilacinus.
35 Conclusion.These pathogenic fungus did not have the risk of pollution or residue, and they can
36 be a alternative option for integrated pest management approache.
37
38 Keywords: Tessaratoma papillosa Drury; entomopathogenic fungi; bioactivity; biological
39 control
40
41
42 43
44
45 Introduction
46 Litchi stink-bug, Tessaratoma papillosa Drury (Hemiptera: Tessaratomidae) is a major pest
47 on litchi and longan in South East Asia and Southern China. The nymphs and adults feed on the
48 tender branchlets, spica and young fruit juice for almost a year, which may be slow growth, wilt,
49 die, and even cause flowers and fruits falling (Waite & Hwang 2002). They had seriously
50 affected the production of litchi and longan. When this insect is disturbed, it may release large
51 quantities of disagreeable acid fluid from its scent gland (Zhao et al, 2012) which causes young
52 leaves wilting and induces brown spots on old leaves and fruits, reducing their edible value and
53 economical value (Bootam & Leksawasdi 1994). Furthermore, it can cause the human eye or
54 skin hurt and inflamed directly (Liu 1965), and also indirectly spread plant viruses, such as
55 witches’ broom disease (Chen et al 2001). It is not only sucking the gramineous plants, but also
56 the virus between crops Chemical pesticide was used in the traditional T. papillosa control ways.
57 However, A huge variety of pesticides not only did not kill the pests, but also resulted in fruit
58 pesticide residues and pest resistance. As a further consequence, It had seriously impact on
59 human health and safety. How to improve the T. papillosa harmless control has become urgent
60 problems of litchi green fruit production (Xu 2005).
61 The egg parasitoid Anastatus spp is mainly used in biological control method of T.
62 papillosa at present (Zou 2008), But there are few reports about other biological control. As an
63 alternative to chemical control or as part of IPM programs, there are many available microbial 64 insecticides resources to utilize for biological control of insect pests. Being a kind of pathogens
65 of agricultural pests, entomopathogenic fungi is a key factor to control insect population density
66 in ecosystem. Many entomopathogenic fungi, such as Metarhizium anisopliae, Beauveria
67 bassiana, Paecilomycesfum osoroseus, Verticillium lecanii, Aschersonia montagne has been
68 developed for the fungus pesticide products, and applied in large biological control of important
69 pests in agriculture and forest. However there are few reports about entomopathogenic fungi of
70 litchi pests cnotrol. In this study, we isolated two pathogenic fungus from the adult diseased T.
71 papillosa by normal methods and rDNA-ITS, and investigate its bioactivities to T. papillosa. We
72 expect to have a certain application valuable bio-control strain of T. papillosa control.
73 Materials and methods
74 Test strain
75 The strains were isolated from the adult of T. apapillosa in litchi orchard, located in
76 Huangwei village, Conghua city, Guangdong Province, China. Good growth and spore
77 production traits of strains were selected as test strains. And they were purified and routine
78 identified according to methods based on the phenotypic properties of the pathogenic fungus
79 morphology (Pu et al 1996, Liang et al 2005, Luangsa-ard et al 2005).
80 Sequencing and phylogenetic analysis of strain ITS
81 Total DNA was extracted according to the manufacturer’s specifications (Omega, E.Z.N.A.
82 Fungal DNA Kit). The DNA specimens were amplified with universal primers ITS1: 5’-
83 TCCGTAGGTGAACCTGCGC-3’, ITS4: 5’-TCCTCCGCTTATTGATATGC-3’and ITS5: 5’- 84 GGAAGTAAAAGTCGTAACAAGG-3’. For the PCR, PCRs were conducted in 25 μL volume
85 containing 0.5 µL Taq DNA polymerase, 2 µL dNTPs, 2.5 µL 10× buffer, 1 µL of each primer, 1
86 µL of total DNA, 17 µL ddH2O (TaKaRa, Japan). Amplification was performed in an Eppendorf
87 Mastercycler ep (Eppendorf, Hamburg, Germany), with PCR-negative controls containing
88 ddH2O instead of template DNA. The amplification profile was as follows: one denaturation step
89 at 94C for 5 min, then 35 cycles of 94C for 30 s, 50C for 30 s, and 72C for 1 min；followed
90 by a final extension at 72C for 5 min. To check the results, 5 L of PCR products were run in a
91 1.0% agarose gel stained with GoldView in 0.5× TBE buffer [45 mmol/L Tris-Cl, 1 mmol/L
92 EDTA] (Sangon, China), and the amplification products were analyzed by comparison with a
93 DNA size marker (TaKaRa, Japan) run in parallel on the same gel. This band was excised from
94 the agarose gel, purified using a DNA gel extraction kit (Omega, USA), cloned into the pMD18-
95 T vector (Takara, Japan), and sequenced completely in both directions (Invitrogen, Guangzhou,
96 China).
97 The sequencing results were analyzed by ‘‘BLAST’’ tools on the National Center for
98 Biotechnology Information (NCBI) website. Then the strains with high homology were
99 contrasted with the test strain. The multiple sequence alignments of deduced amino acid
100 sequences were performed using ClustalW (Thompson et al 2002) multiple-alignment software
101 (http://www.ebi.ac.uk/clustalw/ index.html). The phylogenetic tree was constructed using the
102 MEGA 4 software (Tamura et al 2007).
103 Pathogenicity test of strain
104 Preliminary screening of the test strains pathogenicity in T. apapillosa 105 The purified strains were inoculated and cultivated on petri dish plates for 5 days, the
106 received conidium were used for bioactivity assay in young nymph, old nymph and adult of T.
107 apapillosa respectively. Put these insects into 1×108 conidium /mL spore suspension of the
108 selected strains for 10s respectively, then took them out and dried them, introduced them inside a
109 petri dish (17 cm in diameter and 3.0 cm high). 10 insects per arena, provided with water
110 supplied on soaked cotton, The petri-dishs were then sealed with a plastic wrap to prevent insects
111 from escaping and placed in a climatic chamber at 26 ± 1C, 14: 10 (L: D) photoperiod, and 60–
112 80% RH. The negative controls consisted of arenas with the same densities of T. apapillosa but
113 without any test strains treatment. Each assay was replicated three times for each petri dish
114 insects’ density. We observe infection situation at regular intervals every day, and the dead T.
115 apapillosa were selected and single cultivated. The dead T. apapillosa caused by the two
116 tested strains were calculated the cumulative mortality respectively, if the culture of
117 entomopathogenic fungi was agrees with the test strains (Hu et al. 2007). The corrected
118 mortality was calculated according to the formula of Abbott.
119 Corrected mortality (%) = [(Mortality of treatment group - Mortality of control group) ×100/
120 (100- Mortality of control group)
121 Toxicity determination of the test strains
122 The high virulent strains from preliminary screening test were used for further toxicity
123 determination of young nymph, old nymph and adult T. apapillosa respectively. The conidial
124 suspension of test strains were diluted into 5 concentration gradient respectively by sterile water
125 (1×108、5×107、2.5×107、1.25×107、6.25×106 conidium/mL), and with sterile water as 126 blank control. 10 insects were treated for each concentration，and each assay was replicated
127 three times. Corrected mortality were calculated, and the LC50 was determined by DPS9.50.
128 Results
129 The characteristic of infected T. apapillosa and the results of routine identification
130 We separated and purified two strains (Ta-01,Ta-02) from the adult of T. apapillosa in litchi
131 orchard (Fig. 1) The artificial infection test in adult of T. apapillosa showed that both strains can
132 cause the infected insects became moved slowly and a slight spasm phenomenon after 48~72 hrs.
133 Ta-01 strain infected T. apapillosa can cause some white hypha grown from antenna and inter-
134 segmental membranes of dead T. apapillosa after they had cultured 7 days . And Ta-02 strain can
135 cause infected T. apapillosa stoped their activity and clung tightly to branch of litchi until they
136 became stiffness, died and grown gray mycelium and conidium in metamere and inter-
137 segmental membranes of dead T. apapillosa after 7 days or longer. The performances in routine
138 identification (Fang et al 2001, Li et al 2004, Lu et al 2007) show that the two strains are
139 Paecilomyces spp and Beauveria spp according to reference, respectively.
140 Sequencing of ITS and Phylogenetic analysis
141 The results of rDNA-ITS sequencing of two strains showed that 559 bp and 451 bp of
142 special DNA fragments was sequenced（Fig. 2）. The yielded sequence was compared with the
143 sequences of 18SrDNA accessed in GenBank by BLAST（Fig. 3）. Phylogenetic analysis
144 indicated the obtained sequence Ta-01 shares high homology with Paecilomyces lilacinus (99%),
145 they formed a cluster；and the obtained sequence Ta-02 formed a cluster with Beauveria
146 bassiana, the homologies was 99%. Together with the routine identification and molecular 147 identification, Ta-01 and Ta-02 was proved to be P. lilacinus and B. bassiana.
148 Pathogenicity analysis of strain
149 In this study, P. lilacinus and B. bassiana were isolated and pathogenicity analysised. The
150 bioassay results showed that，the two strains both had good pathogenicity in T. apapillosa. And
151 the higher pathogenicity strain is B. bassiana，the corrected mortality rate of infected young
152 nymph, old nymph and adult T. apapillosa within 10d were 88.89±11.11%, 75.56±7.70%,
153 65.56±5.09% respectively, and they had a greater difference between infected young nymph and
154 adult T. apapillosa by B. bassiana (P>0.05). However, the corrected mortality of infected young
155 nymph, old nymph and adult T. apapillosa by P. lilacinus were 78.15±11.67 %, 68.89±10.18%,
156 63.33±5.77%, and they were no significant difference (P>0.05) (Table 1).
157 The toxicity determination of the two strains showed the LC50 value for B. bassiana infected
158 young nymph and old nymph of T. apapillosa within 10 d were 1.43×107 and 1.36×107, whereas
159 they were higher than P. lilacinus infected different of T. apapillosa (young nymph: 1.92×107 ;
160 high-instar nymph: 1.95×107; adult: 2.54×107 (Table 2).
161 Discussion
162 Research biological control technology of major pest of litchi tree is a basic safeguard to
163 produce pollution-free litchi. T. apapillosa Drury is a major pest on litchi and longan in Southern
164 China. Previous studies pay more attention on utilizing part of natural resources for its control.
165 Especially using the parasitic wasp for controling T. apapillosa and other important litchi pests
166 has made substantial progress (Liu et al 1995, Han et al 1999, Chi et al 2006). The azadirachtin
167 tree injection should also be tested against T. apapillosa and other pests of litchi (Marie Joy 168 Schulte et al 2006). Entomopathogenic fungi was regarded as a vital ecological factor pressing
169 pest populations in field, however, there were few researches conducted on entomopathogenic
170 fungi against T. apapillosa. In our studies, we identified primarily entomopathogenic fungi
171 which we sampled from adult diseased T. papillosa in litchi orchard to be B. bassiana and P.
172 lilacinus. and we confirmed that they also play an important role in the control of T. apapillosa.
173 At present, the approaches of fungal systematics in addition to the traditional
174 morphological identification, the determination of nucleic acid sequence is also needed. One of
175 effective classification identification methods is rDNA-ITS sequencing by PCR。Ta-01 formed
176 a cluster with Paecilomyces spp in NCBI database, and shares 99% high homology with P.
177 lilacinus (KF766523.1); Ta-02 displayed more similar morphological characters in color and size
178 of Beauveria spp , and showed a close relationship supported by a 99% bootstrap value with B.
179 bassiana (JQ991615.1).
180 This study observed that the T. apapillosa infected with B. bassiana and P. lilacinus
181 were initially infected from antenna, metamere and inter-segmental membranes. The results of
182 our pathogenicity analysis of B. bassiana and P. lilacinus showed that the two strains both had
183 good pathogenicity in T. apapillosa, and have a significantly different. The higher pathogenicity
184 strain is B.bassiana, and there are differences mortality rate and death speed with different age of
185 T. apapillosa. The toxicity determination of the two strains showed the LC50 value for B.
186 bassiana was higher than P. lilacinus. These strains showed the potential to control T. apapillosa.
187 They can be developed into commercial preparations for T. apapillosa biological control in litchi
188 orchards when they are trained to have good conidial production levels in fermenter. 189 Entomopathgenic fungi are one of vital microorganisms to control pests and have
190 potentials in biological control. They have the advantages of non-pollution, safety,
191 without pesticide resistance, and they have a longer controling duration during the host
192 reproductive dispersal in pest populations and epidemic caused. However, In the long-
193 term use process of biopesticide, entomopathgenic fungi can result in biological control effect
194 present a slowly insecticidal rate and a unstable phenomenon because of strain degradation,
195 variation, rejuvenation and contamination. The culture and store of B. bassiana and P. lilacinus;
196 the correlation of B. bassiana and P. lilacinus virulence with different age of T. apapillosa,
197 ambient temperature and humidity, and other factors; and the field application and control effect
198 of B. bassiana and P. lilacinus for T. apapillosa control remains to be further research.
199
200 Acknowledgments
201 We are grateful to the members of our laboratory for their cooperation in this work.
202
203
204 ADDITIONAL INFORMATION AND DECLARATIONS
205 Funding
206 This research was supported by Science and Technology Planning Project of Guangdong
207 Province, China (2014A020208079; 2015A020209091); Science and Technology Planning
208 Project of Guangzhou, Guangdong Province, China (201610010002); Guangdong Aacademy of
209 Sciences Outstanding Young and Scientific Talent Foundation (rcjj2015). The funders had no 210 role in study design, data collection and analysis, decision to publish, or preparation of the
211 manuscript.
212 Grant Disclosures
213 The following grant information was disclosed by the authors:
214 Science and Technology Planning Project of Guangdong Province, China: 2014A020208079;
215 2015A020209091;
216 Science and Technology Planning Project of Guangzhou, Guangdong Province, China:
217 201610010002;
218 Guangdong Aacademy of Sciences Outstanding Young and Scientific Talent Foundation:
219 rcjj2015.
220 Competing Interests
221 The authors declare there are no competing interests.
222 Author Contributions
223 ·Xiang Meng conceived and designed the experiments, performed the experiments, analyzed
224 the data, wrote the paper, prepared figures and tables.
225 ·Xiang Meng and Junjie Hu performed the experiments, analyzed the data, contributed
226 reagents/materials/analysis tools.
227 ·Gecheng Ouyang conceived and designed the experiments, wrote the paper, reviewed drafts of
228 the paper.
229 Data Availability
230 The raw data has been supplied as Supplemental Dataset files. 231 Supplemental Information
232 Supplemental information for this article can be found online at
233
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306 Table 1 Virulence of the two strains against the different stages of T. apapillosa 10 d after
307 treatment (Mean±SD)
308
Species Insect stages Sample(N) Corrected mortality(%)
Paecilomyces lilacinus young nymph 10 78.15±11.67 ab
old nymph 10 68.89±10.18 b
adult 10 63.33±5.77 b
Beauveria bassiana young nymph 10 88.89±11.11 a old nymph 10 75.56±7.70 ab
adult 10 65.56±5.09 b
309 Note: Different small letters fllowing the data within a column indicate significant difference at 0.05 level.
310
311 Table 2 Equations of LC50 values of the two strains against the different stages of T.
312 apapillosa 10 d
313
Species Insect stages Toxicity regression equation Correlation coefficient Conidium /mL
young nymph Y=3.4458+1.2116x 0.9825 1.92×107
Paecilomyces lilacinus old nymph Y=3.8768+0.8712x 0.9494 1.95×107
adult Y=3.9036+0.7801x 0.9398 2.54×107
young nymph Y=3.4326+1.3552x 0.9679 1.43×107
Beauveria bassiana old nymph Y=3.9315+0.9437x 0.9725 1.36×107
adult Y=4.0263+0.7394x 0.9532 2.07×107
314 Figure 1. Two pathogenic fungus from the diseased adult of T. apapillosa in litchi orchard.
315 A: A fungus isolate Ta-01; B: A fungus isolate Ta-02. Series 1: Adult of T. apapillosa
316 invaded by fungi; Series 2: Colony of the strain on PDA; Series 3: Conidioma shape and conidia.
A1 B1
A2 B2 A3 B3
317
318 Figure 2. The amplification product of rDNA-ITS gene of strain Ta-01and Ta-02. M:DNA
319 marker; 1: Ta-01; 2: Ta-02.
320
321
322
323 Figure 3. Phylogenetic based on the 28SrDNA sequence of strain Ta-01 and Ta-02 strains.
324 MEGA 4 was used to construct the phylogenetic tree. Bootstrap analyses from 1000 replications
325 are shown by each branch. 326