Controlling Myzus persicae with recombinant endophytic fungi Chaetomium globosum expressing Pinellia ternata agglutinin

using recombinant endophytic fungi to control aphids

Authors


Xiuyun Zhao, State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Shizishanjie 1, Hongshan District, Wuhan 430070, Hubei Province, China. E-mail: xiuyunzh@yahoo.com.cn

Abstract

Aims:  Sap-sucking insect pests have become the major threats to many crops in recent years; however, only a few biopesticides have been developed for controlling those pests. Here, we developed a novel pest management strategy, which uses endophytes to express anti-pest plant lectins.

Methods and Results:  The fungal endophyte of Chaetomium globosum YY-11 with anti-fungal activities was isolated from rape seedlings. Pinellia ternata agglutinin (pta) gene was cloned into YY-11 mediated by Agrobacterium tumefaciens. The positive transformants, as selected by antibiotic resistance, were evaluated using PCR and Western blot assay. We found that the recombinant endophytes colonized most of the crops, and the resistance of rape inoculated with recombinant endophytic fungi significantly inhibited the growth and reproduction of Myzus persicae.

Conclusions:  Our results showed that the recombinant endophytes expressing Pinellia ernata agglutinin (PTA) may endow hosts with resistance against sap-sucking pests.

Significance and Impact of the Study:  This research may have important implications for using endophytes to deliver insecticidal plant lectin proteins to control sap-sucking pests for crop protection.

Introduction

As a common way to control pests, chemical pesticides have been extensively used in agricultural industries for the past several decades, which, however, may cause serious environmental problems (Lacey and Harper 1986). To reduce the use of chemical pesticides, enhancing the crop resistance to insects provides a more environmentally friendly way, which is usually achieved by making transgenic crops with resistance to pests. Resistance genes like δ-endotoxin from Bacillus thuringiensis (Bt toxin) have been used to make pest-resistant transgenic crops in recent years (Gatehouse 2008). However, it is well known that Bt toxin is only effective against Lepidopteran, Dipteran, and Coleopteran, but not against Homopteran (e.g., aphid and planthopper) that has caused severe losses of crop yields around the world (Gatehouse 2008).

Several transgenic crops such as transgenic cotton and rice expressing Bt toxin have already been planted in several countries to control Lepidopterous pests. However, studies showed that the pest population may change if transgenic crops are solely planted (Liu et al. 2005; Cattaneo et al. 2006). As a result, sap-sucking insect pests (e.g., red spiders, aphids, cotton plant bugs and planthoppers) are becoming the main threats to agricultural industries with the increasing plantation of transgenic crops expressing Bt toxin (Liu et al. 2005; Cattaneo et al. 2006). Despite that sap-sucking pests may cause severe losses of transgenic and traditional crops, there is still no effectual biopesticide currently available for controlling these pests.

Recent studies showed that some plant lectins were toxic to sap-sucking insect pests. For example, the lectin from snowdrop (Galanthus nivalis agglutinin) is toxic to planthoppers by binding itself to gut epithelium and then passing into haemolymph of pest (Fitches et al. 2001). The mannose-binding Pinellia ternata agglutinin (pta) gene was also found with significant insecticidal activities against Homopterans (Yao et al. 2003a,b; Zhang et al. 2003). Insect bioassays showed that transgenic plant expressing PTA lectin gene significantly inhibited aphid growth and exhibited considerable insecticidal activities against rice brown planthopper. To the best our knowledge, there are still no other insecticidal proteins, which are as adequately effective as plant lectins to control sap-sucking insects.

Endophytic micro-organisms are those that inhabit in the interiors of plants, especially in leaves, branches, and stems, without apparent harm to the hosts (Azevedo et al. 2000). The capability of colonizing internal host tissues makes endophytes a valuable tool for agricultural industries to improve crop performance. Some endophytic micro-organisms have received considerable attentions in the last two decades because of their capabilities to protect hosts from pests and pathogens. Endophytic fungi are found in the majority of currently known plants and can be isolated from plant tissues after strict surface sterilization. Endophytic fungi can be potentially used for improving plant growth, fixing nitrogen, and particularly biologically controlling pests and plant diseases because of their capabilities to habitat in plants after inoculation (Saikkonen et al. 1998).

In a previous study, we have isolated endophytic fungal strain of YY-11 from rape seedlings and characterized as Chaetomium globosum. The experimental results clearly showed that YY-11 exhibited activities against several phytopathogenic fungi, including Rhizoctonia solani, Botrytis cinerea, Fusarium oxysporum f.sp. Vasinfectum, F. graminearum, Sclerotinia sclerotiorum, and Botryosphaeria berengriana f.sp. piricola (unpublished data). YY-11 could colonize different types of crops, such as, rice, wheat, maize, cotton and radish.

In this study, we used endophytes to deliver and express PTA lectin for developing a novel strategy against sap-sucking pests. Different from transgenic plants, recombinant endophytes can enhance the resistance of the hosts to sap-sucking pests without changing other performances of the hosts. Endophytes usually exist in plants during their growing stages, but are absent in seeds (Azevedo et al. 2000). Therefore, grain crops (e.g., rice, maize, and wheat) inoculated with recombinant endophytes expressing PTA lectin, are relatively safe to consume. We hypothesized that hosts inoculated with recombinant endophytes expressing PTA lectin would be endowed with the resistance to sap-sucking insect pests.

Materials and methods

Endophytes

YY-11 (C. globosum) is an endophytic fungus isolated from rape seedlings in the field at Wuhan City, Hubei Province, China. The colony of YY-11 was with yellow colour, producing yellow spores after incubation on a potato-dextrose agar (PDA) plate at 28°C for 5 days. It can produce ellipse-shaped ascospores. The ascospores were produced within flask-shaped perithecia and not exposed to air. When the spores mature, they are released through an opening at the top of the perithecium.

Construction of vector and transformation of endophyte

Eukaryotic vectors were constructed to respectively express PTA in endophytic fungi. For the endophytic fungi, the pta gene was amplified by PCR from the plasmid of pT-pta containing pta gene (GU593718) (Yao et al. 2003a,b) and then inserted into pCAMBIA1301 plasmid (Yang et al. 2010) to form a recombinant plasmid of pCAMBIA1301-PTA (Fig. 1a). This recombinant plasmid was transformed into Agrobacterium tumefaciens, and positive transformants were used for transforming YY-11 using the methods by (Yang et al. 2010). The transformants were further verified through amplification of the hygromycin resistance gene and pta gene by PCR.

Figure 1.

 Schematic map of recombinant plasmid pCAM1301-PTA. Hygromycin resistance gene (hygromycin R) was used as a marker for screening positive transformant on PDA plate with hygromycin. CaMV35S promoter was used to control expression of lectin and hygromycin resistance genes.

SDS-PAGE analysis, Western blot assay and ELISA

To detect whether PTA was expressed in the recombinant endophytic fungi, the selected positive transformants were further analysed by the Western blot assay. Briefly, YY-11 transformant was frozen using liquid nitrogen and then dissolved with phosphate buffer (pH 7·2). The amount of protein loaded on SDS-PAGE is 50 μg. The supernatant was collected for analysis by 15% SDS-PAGE and then transferred onto nitrocellulose membrane. The membrane was blocked with 2% bovine serum albumin (BSA) at room temperature (RT) and then incubated overnight with mouse anti-PTA serum (reserved in our lab) at 4°C. After being extensively washed by PBST (PBS containing 0·1% Tween 20), the membrane was incubated with goat anti-mouse IgG antibody conjugated with Horseradish Peroxidase (HRP; Sigma, St Louis, MO) and then developed with TMB (3,3′,5,5′-tetramethylbenzidine) for 15 min.

Anti-PTA mouse serum was used for determining recombinant PTA by ELISA. Protein was purified from recombinant C. globosum and diluted in 0·05 mmol l−1 carbonate-bicarbonate buffer (pH 9·6), and then added into ELISA plates (Costar, Bloomington, MN) for incubation at 4°C overnight. After that, plates were blocked with 1% (w/v) bovine serum albumin (BSA; Sigma) at RT for 2 h, and then incubated with 1 : 10 000 dilution of anti-PTA mouse serum at RT for 2 h. After washed five times by PBST (PBS containing 0·05% Tween-20), each well was incubated with 50-μl HRP-conjugated goat anti-mouse IgG (Tiangen, Beijing, China) diluted at 1 : 1000 in PBST at RT for 1 h. Wells were washed five times by PBST and then developed with 50 μl 0·01% 3,3′,5,5′-tetramethylbenzidine (TMB) and 50 μl 0·24% (w/v) H2O2-urea solubilized in 0·2 mol l−1 Na2HPO4– 0·1 mol l−1 citrate buffer (pH 5·5) at RT for 10 min. The reaction was stopped by the addition of 50 μl 2 mol l−1 H2SO4, and then the OD450 value was measured. PTA purified from P. ternate and BSA was used for coating plates as controls for ELISA.

Host specialization of recombinant YY-11

The used wild-type endophytic micro-organisms can colonize different types of crops, such as, rice, wheat, maize, cotton and radish. To detect whether the recombinant endophyte could still colonize in these crops, seedlings of rice, cotton, maize and wheat were cultivated in pots. After two true leaves appeared, each seedling was inoculated with 1-ml ascospore suspension (106 per ml) of the recombinant YY-11 (designated as rYY-11) by pouring on seedling roots. After 100 days of the inoculations, the recombinant endophytic fungi were respectively isolated from seedlings after strict surface sterilization and then verified by PCR through amplifying the pta gene.

Anti-aphid activity of rYY-11 in rape seedlings

To detect whether the recombinant endophytic fungi YY-11 improve crop resistance against aphids, a total of 24 pots of rape seedlings (three seedlings in each pot) were randomly divided into six groups with four pots for each group, and then inoculated with 5-ml ascospore suspension (105 per ml) of rYY-11, wild YY-11, or PBS for each pot. After another 15-day growth, three groups of rape seedlings inoculated with rYY-11, wild YY-11 or PBS were inoculated with 20 third-instar nymphs of aphids (Myzus persicae) for each pot and then covered by an insect-proof net to avoid escape. After that, seedlings were maintained in a greenhouse. The number of survived aphids in each group was counted every day for a total of 25 consecutive days.

To detect whether the recombinant endophytic fungi YY-11 protect rape from aphids, an experiment was carried out to mimic a natural environmental condition. Three groups of rape seedlings inoculated with rYY-11, wild YY-11 or PBS were grown in a greenhouse for 10 days. These seedlings were not covered by an insect-proof net and randomly surrounded by other rape seedlings with aphids. These aphids would naturally move onto the inoculated seedlings. Within a total of 45-day growth under a normal condition, the number of aphids on each rape seedling was counted at day 7, 14, 21, 28, 35 and 45.

Anti-fungal activity of rYY-11

YY-11 exhibited activities against several phytopathogenic fungi. To detect whether the recombinant YY-11 also exhibit activities against phytopathogenic fungi such as S. sclerotiorum, which is an important pathogen of rape, Sclerotinia sclerotiorum was co-cultured with rYY-11 on a glass slide covered by a thin layer of PDA agar at 28°C for 4 days. The interactions between those two fungi were determined by a phase contrast microscope. Rape seedlings (3 weeks old) were grown in pots with three seedlings in each pot and then inoculated with 5-ml ascospore suspension (105 per ml) of rYY-11. After 10 days of the inoculation, 20 mycelial discs (5 mm) of S. sclerotiorum were inoculated on the root and leaf of rape seedlings in each pot. Seedlings were maintained under controlled conditions (25°C and photoperiod of 12 h day−1, and 90% relative humidity) for the development of rape sclerotinia rot. Control plants were inoculated only with PBS buffer ((g l−1) NaCl, 8; KCl, 0·2; Na2HPO4, 1·4; KH2PO4, 0·24) and S. sclerotiorum. The symptoms were evaluated after 15 days of disease development.

Observe YY-11 in stems of rape seedlings under transmission electron microscope (TEM)

The stem of aseptic rape seedlings was cut into pieces with 3 cm in length and put on the both sides of glass slide covered with a thin layer of PDA medium. C. globosum was pre-inoculated on middle of the PDA medium. The distance between stem and hyphae was about 0·3 cm. The slide was put on wet filter paper in a culture dish and then incubated at 25°C without light until the fungus hyphae grown and contacted with the stem. After further cultured for 36 and 48 h, the stem was cut into 0·5 cm of slice and fixed in glutaraldehyde for 4 h in vacuum. After dehydration, the samples were sprayed with aurum after critical point drying and then watched by a TEM (H-7650).

Statistical analysis

Each experiment in this study had three repetitions. The statistical differences among groups were analysed by a one-way analysis of variance (anova) with a Tukey’s post hoc test (spss 16.0; SPSS Inc., Chicago, IL).

Results

Construction of recombinant endophytes

The plasmid pCAMBIA1301 is a vector usually used for transforming a foreign gene into plants and for making transgenic fungi (Yang et al. 2010). The constructed recombinant pCAM1301-PTA was transferred into A. tumefaciens and then used for transferring YY-11. The positive transformants selected by hygromycin resistance were further verified by PCR (Fig. 2a). The total soluble proteins in the transformants were analysed by SDS-PAGE, which showed that PTA was successfully expressed by rYY-11 (recombinant YY-11) with the same molecular weight as native PTA protein extracted from P. ternate corm (Fig. 2b). This protein was then analysed by Western blot, which showed that the protein was recognized by mouse anti-PTA serum (Fig. 2c). These results indicated that PTA was successfully expressed by rYY-11 as a soluble protein. The PTA concentration was determined as 18·5 μg ml−1 by ELISA assay. This proved that high level of PTA protein was expressed by recombinant C. globosum.

Figure 2.

 Analysis of positive transformants C. globosum YY-11. (a) PCR verification of rYY-11. Lane W, wild YY-11; lane M, DL2000 DNA ladder (Takara, Japan); lane 1-5, transformants of YY-11. Arrow indicates the amplified DNA fragment about 750 bp. (b) SDS-PAGE analysis of recombinant C. globosum YY-11. Lane M, standard protein markers (from top to bottom 97, 66, 35, 27, 20, 14·4 kDa); lane P indicated PTA lectin extracted from P. ternata; lane 1-2, total cellular proteins of rYY-11; lane 3-4, total cellular proteins of wild YY-11. (c) Western blot analysis of positive transformant of C. globosum YY-11. Arrow indicates the recombinant PTA protein expressed by rYY-11.

Recombinant endophytes colonizing crops

Like wild types, the recombinant endophytes could also colonize many crops. The results showed that rYY-11 colonized in rape, cotton, rice, wheat, cabbage and radish for more than 1 month (Fig. 3). These results showed that the recombinant endophytic fungi could colonize various types of crops, indicating a potential for controlling pests with piercing-sucking mouthparts on many crops in the future.

Figure 3.

 Colonization of different crops by C. globosum rYY-11. (a) rYY-11 was re-isolated from inoculated plants 1 month postinoculation. (b) PCR screening of plant rYY-11 isolates. Lane M, DL2000 DNA ladder (Takara, Japan); lane 1-4, PCR amplified the pta gene from the YY-11 re-isolated from rape, cotton, rice and wheat, respectively; lane 5-6, positive controls. Arrow indicates the pta gene about 750 bp.

Inoculation of rYY-11 protecting hosts from aphids

Insecticidal activities against aphids were detected for the recombinant YY-11 expressing PTA. rYY-11 was used for the inoculation of rape seedlings. Aphids were then introduced on the seedlings and covered by a net for avoiding insect escape. As shown in Fig. 4a, the number of aphids on the rape seedlings inoculated with rYY-11 was not significantly different from the one of the controls in the first 2 days. However, the number of aphids fed on the rape seedlings inoculated with rYY-11 was significantly different from that inoculated with wild YY-11 and PBS from day 5. The rape seedlings showed an anti-aphid ability after the inoculation with rYY-11. The average number of aphids on the rape seedlings inoculated with rYY-11 was always less than that inoculated with wild YY-11 or PBS before day 25 (P < 0·001). On day 25, the average number of aphids on the seedlings inoculated with rYY-11 was only 27, which was significantly less than that on the seedlings inoculated with wild YY-11 (125 aphids per seedling) (P < 0·0001) or PBS (167 aphids per seedling) (P < 0·0001). This result indicated that the plant was endowed with the anti-aphid ability after inoculated with rYY-11. The number of aphids in the YY-11 group seemed lower than that in the PBS control group, but there was no statistically significant difference between these two groups, indicating that wild YY-11 had no anti-insect activity by itself.

Figure 4.

 Anti-aphid activity of rYY-11. (a) Survival of aphid on rape seedlings inoculated with rYY-11. (b) Average number of aphid on each rape seedling in a mimicry natural environment. (c) aphids on rape leaf in a mimicry natural environment. (inline image) rYY-11; (inline image) WildYY-11; (inline image) Control; (inline image) rYY-11; (inline image) YY-11; (inline image) PBS.

In addition to the ‘no-choice’ tests, in which aphids were not allowed to freely move among plants, we also detected the anti-aphid ability for seedlings in a greenhouse, where aphids could freely move among plants. The result showed that the number of aphids was significantly less on the rape seedlings inoculated with rYY-11 than that inoculated with wild YY-11 or PBS (Fig. 4b). At day 7, aphids were found to move onto plants inoculating rYY-11 and wild YY-11 from the rape seedlings with insects. During this period, the number of aphids was similar among the groups of rYY-11, wild YY-11 and PBS. However, from day 14 until the end of the experiments, aphids on the seedlings inoculated with rYY-11 was significantly less than that inoculated with wild YY-11 or PBS. At day 45, the average number of aphids was 9·2 ± 5·3 (mean ± SD) on the seedlings inoculated with rYY-11, which was significantly less than that inoculated with wild YY-11 (116 ± 13) (P < 0·0001) or that inoculated with PBS (120·7 ± 28·5) (P < 0·0001), indicating that the rape seedlings inoculated with rYY-11 were resistant to aphids (Fig. 4b,c).

Recombinant endophytic fungi antagonizing phytopathogenic fungi

On the basis of microscopic observations, it was found that the hyphae of rYY-11 could attach to and wind around the hyphae of S. sclerotiorum (Fig. 5a), indicating that rYY-11 had an apparent mycoparasitism during the confrontation with S. sclerotiorum, which is consistent with previous studies (Cao et al. 2009). Rape seedlings pre-inoculated with rYY-11 resisted the infection of S. sclerotiorum, while seedlings in the control group were seriously infected and killed by this fungus (Fig. 5b). This result indicated that YY-11 did not lose its anti-fungal activity after being transferred with exogenous genes such as plant lectins.

Figure 5.

 Anti-fungal activity of rYY-11. C. globosum rYY-11 colonized the S. sclerotiorum mycelium and caused deformation (a) and enlargement (b) of infected hyphae. Rape seedlings pre-inoculated with the rYY-11 could also resist the infection of S. sclerotiorum (c).

Colonization of YY-11 in stems of rape seedlings under transmission electron microscope (TEM)

The colonization of C. globosum in stem of rape seedlings was observed under TEM, as shown in the Fig. 6, the hyphae of C. globosum can grow in stem cells of rape seedlings.

Figure 6.

 Colonization of YY-11 in roots of rape seedlings under transmission electron microscope. Hyphae of YY-11 grew in cell of the stems of rape. (a,b) Bar = 2 μm. (c) Bar = 1 μm.

Discussion

For the past several decades, the techniques, such as plant breeding and integrating foreign DNA into plant genomes to make transgenic plants, have been routinely used to enhance plant resistance to pests. However, these methods are generally costly, which may take years to be commercialized depending on plant species. Meanwhile, cloning insecticidal protein genes in transgenic plants provides an alternative way; however, its applications are generally limited in some monocotyledonous plants such as sugarcane, rice, sorghum, and maize. In comparison, beneficial endophytes can be also used to express and secrete useful products, but do not require the integration of foreign DNA into plant genomes. Therefore, this technique is of great use to introduce insecticidal toxin genes into the endophytes with the capability to colonized plants. Here, for the first time, we report the effects of an endophyte recombinant strain on Homopteran. We found that C. globosum YY-11 carrying the P. ternate agglutinin gene was toxic to aphids. The number of aphids on seedlings was greatly suppressed by the endophyte recombinant strain infection. Therefore, plants inoculated with the recombinant endophytic fungi contain plant lectin proteins and gain the resistance to insects.

Many endophytes have mutualistic relationships with their host plants, from which they obtain nutrients and in turn provide protection for the host plants from biotic and abiotic stresses. Alkaloid produced by endophytes can significantly reduce herbivores (Clay 1996; Clay and Schardl 2002; Siegel et al.1990). For instance, some endophytes can produce toxic alkaloids such as Neotyphodium coenophialum, which exist inside Tall fescue (Schedonorus phoenix (Scop.) Holub) and greatly limits animal feeding and reproductive performance. In banana, naturally occurred endophytic Fusarium oxysporum antagonized nematode Radopholus similis in vitro through the production of nematode-antagonistic metabolites (Dubois et al. 2004; Athman et al. 2006). Inoculation of those endophytes into tissue culture plants resulted in improved plant growth and reduced nematode densities (Dubois et al. 2004; Athman et al. 2006). Other benefits of using endophytes may include increased nutrient uptake (Lyons et al. 1990; Malinowski et al. 2000) or enhanced photosynthetic rates (Marks and Clay 1996; Newman et al. 2003). In our study, the endophytic fungi C. globosum YY-11 exhibited no anti-insect activities. Only after transferring the pta gene into endophytes, the recombinant endophytes gained anti-insect activities.

Endophytes colonize an ecological niche similarly to that of the phytopathogenes. This could favour endophytes as agents for the control of pathogenic microbes (Araújo et al. 2001). Some endophytic bacteria or fungi have resistance to phytopathogenic fungi or can promote plant growth, such as C. globosum in this study, which showed an excellent anti-fungal activity. Here, the recombinant C. globosum YY-11 could reduce the infection of phytopathogenic fungi S. sclerotiorum to rape. rYY-11 was able to completely inhibit the development of pathogenic symptoms in the studied seedlings. So, the recombinant C. globosum YY-11 could be used to resist both S. sclerotiorum and aphids.

The results of the anti-insect detection study demonstrate the feasibility of using endophytic fungi, altered by the plasmid vector introduction of an insecticide-coding gene, for controlling an insect pest on a major agricultural crop. Here, anti-insect activities of the recombinant C. globosum YY-11 were similar to that of transgenic tobacco expressing Pinellia ternata agglutinin (Yao et al. 2003a,b), implying that recombinant endophytes could substitute transgenic plants for controlling insects. It is important to note here that before being commercialized, the effectiveness of this technique for controlling aphids still needs to be tested in the field, in which the effect of recombinant endophyte infestation on the yields of inoculated and uninoculated crops can be assessed. The future work involves the construction of strains expressing increased levels of plant lectins that are comparable to current commercial bio-control agents.

The supernatant protein secreted by recombinant endophytic fungi C. globosum was prepared and detected by SDS-PAGE analysis and Western blot to analyse PTA protein. From the result of SDS-PAGE and Western blot analysis, specific protein band reacting with anti-PTA antibody was detected. It was concluded that recombinant endophytic fungi C. globosum can excrete PTA to the outside of cells.

PTA can be expressed and secreted to outside of plant cell in Pinellia ternata. PTA protein has a 24 amino acids signal peptide (Yao et al. 2003a,b), which can lead PTA being secreted outside of plant cell. To express PTA protein at outside of plant cells, the coding sequence and N-terminal signal peptide of PTA lectin gene was cloned into vector pCAMBIA1301. Recombinant vector pCAM1301-PTA was transformed into C. globosum. The T-DNA including CaMV35S promoter and PTA gene was integrated into genomic DNA of C. globosum. When recombinant C. globosum was inoculated to rape seedlings, it can colonize in stem and leaf of seedlings and grow inside or outside of plant cells. C. globosum expressed and secreted PTA protein to outside of plant cell through N-terminal signal peptide of PTA. Secreted PTA protein will go through leaf and stem and reach phloem. When aphid feed on plant and uptake liquid from stem and leaf, PTA will be introduced to the aphids and become toxic against aphids.

Some people try to express PTA protein using micro-organism such as Escherichia coli. P. ternata agglutinin has been expressed as inclusion bodies in E. coli M15 (Lin et al. 2003); people may want to use the recombinant strain to express PTA and control the aphid. But its disadvantage is that the recombinant strain, when sprayed on the surface of plant leaf and stem, was vulnerable to UV degradation and rain washoff. The recombinant protein expressed by these micro-organism will easily to lost their function at the outside environment when encounter UV damage and instable temperature. In contrast to the exposed state of micro-organism following spray operations, PTA expressed by endophytic C. globosum would remain packaged as an intracellular or intercellular protein within the cell, providing protection against UV effects and washoff. Field applications of insecticidal lectin proteins in recombinant C. globosum, therefore, could have the benefit of a longer residual effect. From the standpoint of its ability to colonize plants as endophytes, the modified C. globosum strain would appear to be an ideal host to deliver P. ternata agglutinin insecticidal proteins for crop protection. In conclusion, we recorded experimental evidence that endophytes do play an important role in controlling pest insects. Insecticidal activities of fungal endophytes recombinant strain on Homopteran were recorded in our study.

Similar to plant lectins, some fungal lectins have been reported to possess insecticidal properties. The Sclerotinia sclerotiorum agglutinin (SSA) is a lectin purified from S. sclerotiorum, a soil borne fungus with a wide range of hosts. Feeding assays with the pea aphid (Acyrthosiphon pisum) on an artificial diet containing different concentrations of SSA demonstrated a high mortality caused by this fungal lectin with a median insect toxicity value (LC50) of 66 μg ml−1. (Hamshou et al. 2010). In our report, the PTA concentration produced by recombinant endophytic fungi C. globosum was determined as 18·5 μg ml−1 by ELISA. After 5-day feeding on rape seedlings inoculated with recombinant C. globosum, the aphid mortality was 39·4%. At day 13, the aphid mortality was 61%. The result showed that C. globosum have the potential to control pest insects such as aphid. The difference between S. sclerotiorum and C. globosum is that S. sclerotiorum could produce lectin protein by itself, but C. globosum was a recombinant fungi with PTA lectin gene integrated in its genome. Both fungi could inhibit the reproduction and survival of aphid and have high toxic against aphid. PTA expressed by C. globosum showed stronger activity and active at lower concentrations. But the effects of SSA were detected more rapidly, after 3 days with SSA instead of after 13 days in case of PTA expressed by C. globosum; this may because artificial diet containing SSA was more quickly and directly toxic to aphid than PTA expressed by C. globosum in plants. Lectins of S. sclerotiorum and C. globosum both have the potential to play an important role in the development of integrate pest management strategies to control pest insects.

Endophytes enter plant tissues primarily through root; however, above-ground portions of plants, such as flowers, stems, and cotyledons, may also be used for entry (Kobayashi and Palumbo 2000). Specifically, the bacteria enter tissues via germinating radicles, secondary roots, stomates or as a result of foliar damage. Here, we found that YY-11 can enter plant tissues through the root (N.L., G.Q. and X.Z. unpublished data). Hyphae of C. globosum can enter plant through interspace between root cells.

Endophytic fungi are potential candidates for systematic delivery of biopesticides to a host plant without direct manipulation of the plant genome. Endophytes can reproduce in plant tissues and expressed plant lectin protein in plant tissue including stem, leaf and roots, which can improve anti-insect activity of plants. Control of Homopteran was achieved by the expression of the plant lectin gene in the endophyte. This method may be used to express more useful proteins in the future. In conclusion, our research confirmed that endophytic fungi could be used as a delivery and expressing lectin against pests with piercing-sucking mouthparts. This research has important implications for controlling sap-sucking pests using a genetically modified endophyte as a biocontrol agent.

Conclusion

This study allowed assessing insecticidal activity of recombinant endophytic fungi expressing P. ternata agglutinin in Homoptera species, aphid. From the present results, it is concluded that recombinant endophytes have obviously insecticidal activity on aphid. Population of aphids was smaller in plant infected with recombinant endophytic fungi than in wild-type endophytic fungi-treated plant and controls. This has approved endophytes are good expressing system for delivery insecticidal protein to plant and enhance resistance of plant to insect.

Acknowledgement

This work was supported by National High Technology Research and Development Program of China (2006AA 10A 210).

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