f u n g a l b i o l o g y 1 1 4 ( 2 0 1 0 ) 3 4 – 4 0

journa l homepage : www. e l sev ier . com/ loca te / funb io

Entomotoxic effects of fungal lectin from Rhizoctonia solanitowards Spodoptera littoralis

M. HAMSHOUa,b, E. J. M. VAN DAMMEb,*, G. SMAGGHEa

aLaboratory of Agrozoology, Department of Crop Protection, Faculty of Bioscience Engineering, Ghent University,

Coupure links 653, B-9000 Ghent, BelgiumbLaboratory of Biochemistry and Glycobiology, Department of Molecular Biotechnology, Faculty of Bioscience Engineering,

Ghent University, Coupure links 653, B-9000 Ghent, Belgium

a r t i c l e i n f o

Article history:

Received 1 June 2009

Received in revised form

6 October 2009

Accepted 9 October 2009

Available online 27 October 2009

Corresponding Editor: Anna Rosling

Keywords:

Biocontrol

Fungal lectin

Lepidoptera

Rhizoctonia solani

Spodoptera littoralis

* Corresponding author. Tel.: þ32 92646086; fE-mail address: ElsJM.VanDamme@UGen

0953-7562/$ – see front matter ª 2009 The Bdoi:10.1016/j.mycres.2009.10.002

a b s t r a c t

The effects of the Rhizoctonia solani lectin (RSA) on the growth, development and survival of

an economically important caterpillar in agriculture and horticulture, the cotton leafworm,

Spodoptera littoralis were studied. The high lectin concentration present in the sclerotes of

the soil pathogen R. solani allowed the purification of large amounts of the pure lectin for

feeding experiments with cotton leafworm. Rearing of insects on a diet containing different

concentrations of RSA exerted a strong effect on the larval weight gain. This effect was

visible at the lowest concentration of 0.1 % RSA at day 8 and day 11. Interestingly with 1

% RSA, there was a dramatic reduction in larval weight of 89 % at the end of L6 which

was followed by a high mortality rate of 82 % in the treated larvae. Furthermore, the other

developmental stages of pupation and adult formation were also affected. In addition, the

data demonstrated that the combination of RSA with Bt toxin yielded synergistic effects.

For instance, 0.03 % RSAþ 0.005 % Bt toxin caused reduced growth rate and higher mortal-

ities. These findings suggest that RSA is an interesting tool that can be used for bioengi-

neering insect resistance in important agronomical crops.

ª 2009 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Introduction domain, which binds reversibly to specific mono- or oligo-

Problems associated with widespread insecticide usage,

together with the development of insect resistance to Bacil-

lus thuringiensis (Bt) toxins in genetically engineered crops,

have resulted in a greater interest of scientists to exploit

the potential of using plant defensive proteins, such as

lectins, to help in combating crop damage. Lectins are a ubiq-

uitous group of proteins and several hundreds of these

molecules have been isolated so far from plants, viruses,

bacteria, fungi, invertebrates and vertebrates including

mammals (Carlini & Grossi-de-Sa 2002). Plant lectins are

defined as proteins possessing at least one non-catalytic

ax: þ32 92646219.t.beritish Mycological Society

saccharides (Van Damme et al. 2008). One of the roles attrib-

uted to plant lectins is their involvement in plant defense

against pathogens and phytophagous insects (Peumans &

Van Damme 1995). This protective activity is in accordance

with the observation that most plant lectins are not targeted

against plant carbohydrates, but preferentially bind foreign

glycans (Peumans et al. 2000). Next to plants, it is of great in-

terest that also mushrooms as well as other non-fruiting

body forming fungi contain lectins. Although many carbohy-

drate-binding proteins from fungi have been reported, very

little is known with respect to their physiological role

(Wang et al. 1998).

. Published by Elsevier Ltd. All rights reserved.

Entomotoxic effects of Rhizoctonia solani lectin 35

The Rhizoctonia solani agglutinin, abbreviated as RSA, is

a lectin that is synthesized by the soil pathogen R. solani (Class:

Basidiomycetes; Order: Polyporales). This plant pathogenic

fungus has an asexual life cycle and survives as vegetative

mycelium and sclerotia. These sclerotia enable the fungus to

survive in the soil under harsh conditions. In 1987, Vranken

and coworkers first reported the purification and characterisa-

tion of RSA. This lectin is a homodimeric protein composed of

15.5 kDa subunits that show high affinity for N-acetylgalactos-

amine (GalNAc) and more complex glycoproteins (Vranken

et al. 1987). It was shown that high concentrations of the lectin

accumulate in the sclerotes (2–3 % of the total soluble protein),

whereas the lectin concentration in the mycelium is usually

rather low (0.1–2 % of the total soluble protein) (Hamshou

et al. 2007). At present, the complete RSA sequence is not

known. However, judging from the N-terminal sequence of

60 amino acids, it can be predicted that RSA shows no impor-

tant sequence homology to other known lectin sequences

(Candy et al. 2001), making this lectin a potentially highly

novel compound for research into innovative methods for

the control of insect pests.

Because of the high lectin concentration in the sclerotes, it

has been suggested that RSA could play a role as a storage pro-

tein and could be involved in the defense of the fungus against

predators (Kellens & Peumans 1990). This hypothesis was put

forward based on some striking similarities with plant lectins

that have a dual role. Indeed, for many highly abundant plant

lectins, it was shown that they combine a role in storage with

a role in plant defense whenever the plant is under attack by

predators (Peumans & Van Damme 1995). Indeed, many plant

lectins have been shown to have toxic effects towards insects

(Van Damme 2008; Vandenborre et al. 2009). Using experi-

ments in which purified lectins were added to an artificial

diet or transgenic plants were used to overexpress a lectin

gene it was clearly shown that carbohydrate-binding proteins

interfere with the growth and reproduction of insects from

different orders. Although evidence shows that the carbohy-

drate-binding activity of plant lectins is necessary for their in-

secticidal activity, the mode of action of lectins in insects

remains enigmatic. Over the last decade lectins particularly

those binding mannose, have received significant attention,

predominantly Galanthus nivalis agglutinin (GNA) (Sauvion et

al. 1996; Down et al. 1996; Couty et al. 2001). Since then the po-

tential use of mannose-binding lectins in plant protection

against several insects has been investigated in detail (Van

Damme 2008; Vandenborre et al. 2009). In addition, some re-

ports have shown that the combination of two different insec-

ticidal proteins in a single system provides an effective insect

control and also reduces the potential for development of

resistant insects. For instance, the combination of GNA with

Bt toxin resulted in synergistic effects (Maqbool et al. 2001;

Zhang et al. 2007).

The present paper reports the effects of RSA delivered via

artificial diet on the survival and growth of the cotton leaf-

worm Spodoptera littoralis (Order: Lepidoptera; Family: Noctui-

dae). This polyphagous noctuid species is an economically

important caterpillar in agriculture and horticulture, damag-

ing at least 87 economically important plant species belonging

to 40 families distributed worldwide (Smagghe & Degheele

1994; Sadek 2001).

Materials and methods

Isolation of RSA

Rhizoctonia solani strain AG 1-1B was grown on autoclaved

wheat grains. To produce large quantities of sclerotia, 25 g of

wheat kernels and 60 ml of water were mixed in 250 ml Erlen-

meyer flasks. After autoclaving, small pieces of approximately

1 cm2 agar covered with mycelium from a 5-d old culture of

R. solani grown on potato dextrose agar were added and the

fungal cultures were incubated in a growth chamber at a tem-

perature of 25–27 �C. After 4–5 weeks the sclerotia were

harvested and used for lectin extraction. Sclerotia were lyoph-

ilized and ground to a fine powder using a coffee mill. The dry

powder was extracted in phosphate buffered saline (PBS,

25 ml g�1 dry�1 weight material) for approximately 12 h at

room temperature. Then the mixture was centrifuged at

3000 g for 10 min and remaining debris removed by passing

the supernatant through filter paper (Whatmann 3MM). Affin-

ity chromatography was performed on a galactose column

equilibrated with PBS. After loading the extract, the affinity

column was washed with PBS until the absorbance of the

effluent at 280 nm was <0.1. Subsequently, the lectin bound

to the column was eluted with 20 mM 1,3-diaminopropane

(DAP). The lectin fractions obtained after the first affinity chro-

matography were brought to pH 7.0 and run on the galactose

column for a second time. The RSA preparation obtained after

the second affinity chromatography was loaded on an anion

exchange chromatography column of Q Fast Flow, equili-

brated with DAP. After washing with DAP the lectin was eluted

using 0.1 M Tris–HCl (pH 7.0) containing 0.5 M NaCl. If neces-

sary, these chromatography steps on the galactose and Q

Fast Flow columns were repeated in order to obtain high-

purity lectin preparations. Finally, the lectin fractions were

dialyzed against water and lyophilized. The purity of the lectin

was analyzed by SDS-PAGE.

Insects

An established colony of the cotton leafworm Spodoptera littor-

alis was reared under standard conditions of 23–25 �C, 60–70 %

relative humidity and a 16:8 (light:dark) photoperiod in the

Laboratory of Agrozoology at Ghent University as described

before (Lemeire et al. 2008). Larvae were fed on artificial diet

(Stonefly Heliothis diet, Ward’s Natural Science, Rochester,

NY), an artificial diet for Lepidopteran larval insects that can

be prepared by adding cold water. Under these conditions,

the duration of each of the first five larval instars is about

three days each, whereas the sixth and last larval stage (L6)

takes approximately six days. Larval instars were determined

on the basis of their respective head capsule width.

Effects of RSA feeding on insect survival, growth anddevelopment

Newborn (0–6 h) first instar larvae of Spodoptera littoralis were

selected from the continuous stock colony and RSA was fed

using Stonefly Heliothis diet. Based on previous range finding

tests, RSA was mixed at three concentrations of 0.1, 0.5 and

36 M. Hamshou et al.

1 % (w/w) during the preparation of the diet. Controls were

fed with untreated diet. The experiment was setup in a 24-

well plate and about 0.75 g of the diet was added to each

well, and ten first instar larvae were transferred to each

well. Fresh diet was prepared every 4–5 d and stored at 4 �C

in airtight containers. After 10 d the larvae were transferred

to 9-mm Petri dishes. At different time points during the ex-

periment the weights of all larvae were monitored. The fresh

weight of the resulting pupae and adults was determined on

the first day following pupation and upon adult eclosion, re-

spectively. Per treatment, three replications of 10 insects

each were performed. The experiment was done twice.

Hence, a total of 60 insects were analyzed for each lectin

concentration.

Effect of RSA combined with Bt toxin

In a similar experimental setup we evaluated the combinatorial

effect of RSA and the Bt preparation Delfin (32 000 UI mg�1, San-

doz, Brussels, Belgium). Three concentrations of RSA (0.005,

0.01 and 0.03 %) and Bt toxin (0.005, 0.01 and 0.03 %) were tested

together with five combinations of RSA and Bt toxin (0.005 %

RSAþ 0.005 % Bt, 0.03 % RSAþ 0.005 % Bt, 0.01 % RSAþ 0.01 %

Bt, 0.03 % RSAþ 0.01 % Bt, and 0.03 % RSAþ 0.03 % Bt). At

different time points during the experiment the larval weight

as well as the percentages of mortality and growth retardation

were determined. For each treatment, three replicates of 10

insects each were performed, and the experiment was done

twice. A total of 60 insects were monitored for each treatment.

Statistical analysis

For each treatment and control, data on reduction in weight

gain of the larvae, pupae and adults are expressed as

means� SEM. To detect significant differences between treat-

ments, data were analyzed by one-way analysis of variance

(ANOVA) and then means� SEM were separated using

a post-hoc Tukey–Kramer test ( p¼ 0.05) in SPSS v15.0 (SPSS

Inc., Chicago, IL). Medium toxicity concentration LC50 and me-

dium effect period LT50 and corresponding 95 % confidence in-

tervals were calculated using GraphPad Prism v4 (GraphPad

Software, San Diego, CA); the goodness that the data fit to

the probit curve model was evaluated based on R2 values.

Fig 1 – Concentration-dependent effect of the fungal lectin

RSA against larval growth and development of the cotton

leafworm Spodoptera littoralis. RSA caused a strong reduc-

tion of larval weight after 8 d (A) and 11 d (B) of feeding on

treated diet as compared to controls. Data are presented as

mean fresh larval weight ± SEM; the numbers of insects per

treatment are given with the data. ANOVA resulted in 4

groups for ‘A’ (F [ 58.529, df [ 23, p< 0.001), in 3 groups for

‘B’ (F [ 94.131, df [ 23, p< 0.001). Values per graphic that

are followed by a different letter (a–d) are significantly

different ( post-hoc Tukey–Kramer test with p [ 0.05).

Results

Effects of RSA feeding on insect survival, growth anddevelopment

The high lectin concentration present in the sclerotes of Rhi-

zoctonia solani allowed the purification of sufficient amounts

of the pure lectin for testing of its insecticidal activity. The ef-

fect of the RSA delivered via artificial diet, on survival, growth

and development of caterpillars was followed for first instar

larvae of Spodoptera littoralis until they became pupa and

then adult. Insect survival was evaluated to analyze the toxic-

ity potential of the lectin, and in addition, weight gain of the

larvae, pupae and adults, as well as percentage and time of

pupation were determined as biological endpoints to investi-

gate lectin effects on insect growth and development.

As shown in Fig 1A and B, RSA exerted a strong effect on

the larval weight gain and this effect was clearly concentra-

tion-dependent. At day 8, the weight of S. littoralis larvae fed

on a diet containing 1 % RSA was dramatically ( p< 0.01) re-

duced with 81� 4 %. The two lower lectin concentrations of

0.1 % and 0.5 % RSA also significantly ( p< 0.05) reduced larval

weight by 34� 6 % and 60� 5 %, respectively, compared to the

controls. As a result of the lectin effects on larval develop-

ment, the majority of the population of the insects treated

with RSA at 0.1 % (44 %, 23/52), 0.5 % (60 %, 33/55) and 1 %

(64 %, 36/56) were in the third instar, while the majority of

the control insects had developed into fifth instar (32 %, 19/

60) and some into sixth instar larvae (12 %, 7/60). With 1 %

RSA, none of the insects had entered in the fifth instar. Later

on at day 11, these entomotoxic effects were maintained

with a respective significant ( p< 0.05) reduction of larval

weights by 34� 7 %, 74� 4 % and 89� 3 % for treatments

with 0.1, 0.5 and 1 % RSA (Fig 1B). This reduction in larval

weight was accompanied with a dramatic retardation of

Entomotoxic effects of Rhizoctonia solani lectin 37

development as the majority of the population was stuck in

the fourth instar (70 %, 38/54) with 1 % RSA and in the fifth in-

star (75 %, 39/52) with 0.5 % RSA. With 0.1 % RSA, 30 % (15/50)

and 70 % (35/50) were in the fifth and sixth instar, while in the

control groups nearly all insects (87 %, 52/60) were already L6

larvae. The latter observations demonstrate that the entomo-

toxic effects caused by RSA could not be counteracted by the

intoxicated larvae. With the use of sigmoid regression, the

LT50, which is the time of feeding treated diet needed to re-

duce the larval weight by 50 %, could be determined, being

7.8 d for 0.5 % RSA (95 % confidence interval of 6.4–9.3 d;

R2¼ 0.78) and 7.4 d for 1 % RSA (95 % confidence interval of

6.4–8.3 d; R2¼ 0.97). Finally, most of these intoxicated insects

died. With 1 % RSA, 82� 3 % of the treated larvae were killed

and failed to reach the pupal stage, and with 0.1 % and 0.5 %

RSA the larval mortality reached about 28 % (Fig 2). To confirm

the entomotoxic potential for RSA, sigmoid regression esti-

mated an LC50 value of 0.32 % (95 % confidence inter-

val¼ 0.11–0.95 %; R2¼ 0.87), that is the concentration of RSA

in the diet needed to kill 50 % of S. littoralis larvae.

All individuals that survived and developed into pupae

showed entomotoxic effects of the accumulated RSA. First,

there was a clear effect on insect development with a strong

retardation of 67� 9 % ( p< 0.0001) with 1 % RSA as the devel-

opmental time from neonate (L1) to pupation took 27.3� 0.4 d

as compared with 16.5� 1.3 d in the controls. The retardation

effect ( p¼ 0.001) was 39.3� 7.7 % when RSA was dosed at 0.5

% in the diet. Second, the surviving pupae were negatively af-

fected as they were smaller in size and weight (Fig 3A and B).

With 1 % RSA, the surviving pupae represented only 18 % (11/60)

of the total number of treated larvae. They were smaller in

size and weight with a reduction of 39� 3 % ( p< 0.05). With

0.5 % RSA, the percentage of successful pupation was only

37 % (44/60) and their pupal weight was significantly

( p< 0.05) reduced by 22� 5 % (Fig 3B). In addition, lethal

Fig 2 – Concentration-dependent effect for induction of

larval and pupal mortality in the cotton leafworm Spodop-

tera littoralis by different concentrations of RSA when fed via

treated diet. Mortality percentages ± SEM are based on two

repeated experiments, each consisting of 30 insects per

concentration; a total of 60 larvae were treated per lectin

concentration. ANOVA resulted in 3 groups (F [ 93.704,

df [ 3, p< 0.0001). Values per graphic that are followed by

a different letter (a–c) are significantly different ( post-hoc

Tukey–Kramer test with p [ 0.05).

Fig 3 – Concentration-dependent effect of the fungal lectin

RSA against pupal and adult growth and development of the

cotton leafworm Spodoptera littoralis resulting from larvae

fed on treated diet. RSA caused a marked reduction of the

weight of the resulting pupae (A) and adults (D) as compared

to controls. Panel (B) shows a pupa with reduced size in the

treatment with 0.5 % RSA (left) compared to a normal pupa

(right) in the control, and (C) a lethal larva–pupa intermedi-

ate. Data are presented as mean fresh larval weight ± SEM

compared to the control; the number of insects per treat-

ment is given with the data. ANOVA resulted in 3 groups for

‘A’ (F [ 19.300, df [ 22, p< 0.001), in 3 groups for ‘D’

(F [ 27.643, df [ 22, p< 0.001). Values per graphic that are

followed by a different letter (a–c) are significantly different

( post-hoc Tukey–Kramer test with p [ 0.05).

larval–pupal intermediates were observed in 4 % of the insects

treated with 0.1 % and 0.5 % RSA (Fig 3C).

At the end of the experiment and with completion of the

insect life cycle from the first larval instar to the adult stage,

21 % (9/43) and 27 % (12/44) of the pupae that had developed

38 M. Hamshou et al.

from larvae fed on 0.1 and 0.5 % RSA, respectively, died before

reaching the adult stage. In contrast there was 92 % successful

development into the adult stage in the control (55/60). Hence

due to the lower pupal weight in the RSA treatments, the

resulting adults were smaller in size and weight (Fig 3D).

The weight of the adults was significantly ( p< 0.05) reduced

by 28� 5 % and 44� 2 % with RSA at 0.5 % and 1 %, respec-

tively. In addition, we observed lethal pupa–adult intermedi-

ates in 4 % of the pupae in the treatments with 0.1 % and 0.5 %

RSA.

Effects of RSA combined with Bt toxin

Combinations of RSA and Bt toxin showed higher entomotoxic

effects against the larval weight than with Bt toxin alone.

When the diet was supplemented with low concentrations

of 0.005, 0.01 and 0.03 % RSA and fed for 8 d, the negative effect

on larval weights was 7� 6 %, 21� 10 % and 25� 11 %, respec-

tively. With Bt toxin at 0.005, 0.01 and 0.03 % this was 12� 6 %,

35� 3 % and 79� 5 %. Typically when 0.005 % Bt toxin was

combined with 0.03 % RSA the entomotoxic effect yielded

49� 1 %, demonstrating an increase in entomotoxic effect of

4-fold over Bt toxin alone. At higher concentrations of RSA

and Bt toxin the negative larval effect was also visible. Com-

bining 0.01 % RSA with 0.01 % Bt toxin inhibited to a higher

extent ( p< 0.05), the larval weight reduction being 67� 2 %.

At higher doses of Bt toxin (0.03 %), there was no increased

effect when combined with RSA (0.005, 0.001 and 0.03 %). For

example, 0.03 % Bt toxin combined with 0.03 % RSA caused

74� 4 % weight reduction which was equal ( p> 0.05) to 0.03 %

Bt toxin alone.

Moreover, addition of 0.005 and 0.01 % of Bt toxin to differ-

ent concentrations of RSA (0.005, 0.01 and 0.03 %) resulted in

an increased mortality of treated larvae of 1.5–2 fold over

that with the Bt toxin alone. For instance, the mortality at

day 11 of treatment increased to 32 % with 0.03 % RSAþ 0.01

% Bt, while the mortality was only 10 % and 16 % when RSA

and Bt were dosed alone in the diet. There was no increase

in mortality when the higher concentration of 0.03 % Bt toxin

was combined with different concentrations of RSA (0.005,

0.001 and 0.03 %).

Discussion

In 1996, Mier and coworkers extensively screened 175 differ-

ent species of fungi for their activity against insects such as

Drosophila melanogaster and Spodoptera littoralis. Therefore,

powdered fungi were added to the rearing medium of both in-

sects and the development of the progeny was monitored.

About 45 % of the fungi (79 fungi out of 175) were shown to

be toxic to D. melanogaster, with an LC100 ranging between

0.1 and 60 mg ml�1. Most of the mushrooms that were highly

toxic to D. melanogaster were also toxic to S. littoralis but in

most cases the LD100 was higher (Mier et al. 1996). These re-

sults indicated that many fungi possibly contain interesting

proteins and secondary metabolites with insecticidal activity.

One group of potentially interesting proteins is the group of

lectins found in many fungi. Unfortunately, although many

fungal lectins have been identified and characterized in

some detail, very little is known with respect to their biologi-

cal activities and their function in fungi. Until now only very

few reports have considered the insecticidal activity of puri-

fied fungal lectins. Trigueros et al. (2003) reported the insecti-

cidal activity of XCL, a lectin from the edible mushroom

Xerocomus chrysenteron (Class: Basidiomycetes, Order: Bole-

tales) towards the aphid Acyrthosiphon pisum (Hemiptera) and

D. melanogaster (Diptera). More recently a more extensive

study of the effects of XCL on the aphid Myzus persicae was

performed, confirming the effect of the lectin on aphid devel-

opment and mortality (Jaber et al. 2007). To our knowledge,

there are no reports dealing with the effects of XCL on

caterpillars.

The present study provides a detailed analysis of the ef-

fects of RSA, a fungal lectin from the pathogen Rhizoctonia sol-

ani against the cotton leafworm S. littoralis. Feeding S. littoralis

larvae on an artificial diet containing different concentrations

of RSA resulted in a significant reduction in larval weight. The

reduction in larval weight was about 89 % when the larvae

were fed on a diet containing 1 % RSA, and 74 % with 0.5 %

RSA for 11 d. Due to the lower weight of the larvae that devel-

oped on the artificial diet containing RSA, the resulting pupae

and adults were smaller in size. Since RSA adversely affected

the weight gain in the larval stage, the development into pu-

pae was also retarded. It is a well known phenomenon that fi-

nal instar larvae have to reach a minimum critical fresh

weight before entering the pupal stage (Davidowitz et al.

2003). As a consequence, RSA caused about 81 % larval mortal-

ity when tested at a concentration of 1 %. So feeding the diet

with RSA to S. littoralis larvae resulted in a clear reduction in

larval weight and a significant larval mortality. A good exam-

ple for comparison of insecticidal potential is the mannose-

binding lectin GNA. Fitches et al. (1997) reported a 32 % and

23 % reduction in the larval biomass of the tomato moth (Laca-

nobia oleracea) fed on an artificial diet containing 2 % GNA or

excised leaves of transgenic potato expressing GNA at 0.07 %

of total soluble protein, respectively. In addition, these au-

thors observed that GNA retarded larval development and af-

fected pupation. However, a significant effect of GNA on larval

survival was only recorded in a glasshouse experiment using

transgenic potato plants (with GNA expression at 0.6 % of total

soluble protein), which resulted in a decrease in larval survival

of approximately 40 %. Using a similar bioassay as the one

used in this study with RSA, Sadeghi et al. (2009) recently

reported that feeding of larvae of S. littoralis with tobacco

leaves expressing another mannose-binding lectin from leek

(Allium porrum) APA at 0.7 %, reduced the larval weight gain

by 15–27 %, and 28 % of the intoxicated larvae were killed be-

fore pupation. Although these results also show clear effects

of the mannose-binding lectins on caterpillar development,

the percentages of weight reduction and mortality are much

lower than observed in this study for RSA. Therefore the

results reported here demonstrate the high potential of the

fungal lectin RSA in the control of important pest Lepidoptera.

Currently, XCL and RSA are the only two known fungal lec-

tins purified from species belonging to the order of Basidiomy-

cetes, and these clearly exhibit specificity for Gal/GalNAc

residues. However, both lectins are not related evolutionary.

The XCL sequence shows significant sequence homology to

the group of Agaricus bisporus related lectins (Trigueros et al.

Entomotoxic effects of Rhizoctonia solani lectin 39

2003), whereas the N-terminal 60 amino acid sequence of RSA

shows some residual sequence similarity to the ricin-B homo-

logs (Candy et al. 2001).

Insecticidal activity of plant lectins has been studied in de-

tail especially for mannose-binding lectins (Van Damme 2008;

Vandenborre et al. 2009). At present the insecticidal properties

of galactose-binding lectins are far less documented. Only

a few galactose-binding lectins were reported to have toxic

effects against caterpillars. For instance, the Bauhinia purpurea

lectin and the Ricinus communis agglutinin revealed entomo-

toxic properties against Ostrinia nubilalis (Czapla & Lang

1990), whereas peanut (Arachis hypogeae) lectin showed activ-

ity against Helicoverpa armigera (Shukla et al. 2005). More

recently, the Annona coriacea lectin was shown to be active

against larvae of two pyralid moths, Ephestia (¼Anagasta) kueh-

niella and Corcyra cephalonica (Coelho et al. 2007). Bauhinia

monandra leaf lectin was toxic to A. kuehniella (Macedo et al.

2007). Similar to our results with the fungal lectin RSA, all

these studies report entomotoxic effects by galactose-binding

plant lectins on insect survival, growth and development. As

already mentioned above, RSA is probably distantly related

to the B-chain of ricin and other type-2 ribosome-inactivating

proteins (Candy et al. 2001). In a recent report, Shahidi-

Noghabi et al. (2009) have shown that treatment of caterpillars

of the beet armyworm (Spodoptera exigua) with the type-2 ribo-

some-inactivating protein SNA-I from elderberry (Sambucus

nigra) resulted in a significant reduction in fresh weight, retar-

dation in development as well as a significant increase in

mortality of S. exigua larvae fed on transgenic lines as com-

pared to wild type plants. Furthermore these authors have

shown that the carbohydrate-binding properties of the elder-

berry lectins are a determining factor in their insecticidal

properties, since mutant lectins that have lost their ability to

recognize carbohydrate structures also loose their insecticidal

activity (Shahidi-Noghabi et al. 2008).

Finally, we also tested the effect of RSA in combination

with a Bt toxin preparation (Delfin). The results show a greater

reduction in larval weight when low concentrations of RSA

and Bt toxins were used. However, at a higher concentration

(0.03 %) of RSA and Bt toxin this combinatorial effect was

less clear and the effect was almost equal to that of the Bt

treatment alone. Delfin contains Cry1Aa, Cry1Ab, Cry1Ac

and Cry2Aa proteins (Singh et al. 2008). Previously it was

shown that the Cry1Ac toxin specifically recognizes GalNAc

and has a binding pocket for this sugar (Burton et al. 1999).

CryAc1 was shown to bind to a specific receptor in the lepidop-

teran gut, namely aminopeptidase N (Hakim et al. in press).

This binding can be inhibited by GalNAc (Gill et al. 1995). How-

ever, the toxicity of Cry1Ac is not solely dependent on GalNAc-

mediated binding (Masson et al. 1995; Banks et al. 2001), and

therefore it seems likely that Bt toxin will bind a broader range

of putative target proteins in the insect midgut than RSA.

These results demonstrated that lectins like RSA can be com-

bined with Bt toxin without impairing insecticidal activity. In

addition, it was of great interest that synergistic activities

were observed when RSA was tested in combination with Bt

toxin in the lower concentration range.

The entomotoxic activity of RSA in diet is a good indication

that this fungal lectin can be a useful tool for bioengineering

insect resistance in crops of agronomic importance. Clearly

the carbohydrate specificity of this lectin is very different

from the mannose-binding lectins which have been studied

in detail for their insecticidal properties. Therefore, it can be

envisaged that RSA will target other proteins in the insect

body, allowing to control important and resistant pest insects.

In addition, our data support that RSA can be combined with

Bt toxin. Genetic engineering of plants for the expression of

two lectins with different carbohydrate-binding specificity

could enable to target a broader range of receptor proteins. It

was already shown that transgenic lines expressing GNA in

combination with Bt toxin (CrylAc and Cry2A) showed greater

reduction in insect survival and greater reduction in plant

damage compared with single treatments (Maqbool et al.

2001; Zhang et al. 2007). Similarly, engineering of plants with

a fusion protein combining the Cry1Ac toxin with the galac-

tose-binding domain of the nontoxic ricin B-chain resulted

in a stronger activity of the fusion proteins against insects

than for Cry1Ac alone (Mehlo et al. 2005).

Acknowledgements

M. Hamshou acknowledges a PhD scholarship from the

Ministry of Higher Education and General Commission for

Scientific Agricultural Research in Syria. This research is also

supported by project 3G016306 to G. Smagghe and E.J.M. Van

Damme from the Fund of Scientific Research (FWO-Vlaande-

ren, Brussels, Belgium). Rhizoctonia solani strain AG 1-1B was

obtained from M. Hofte, Laboratory of Plant Pathology and

Virology, Ghent University.

r e f e r e n c e s

Banks DJ, Jurat-Fuentes JL, Dean DH, Adang MJ, 2001. Bacillusthuringiensis Cry1Ac and Cry1Fa d-endotoxin binding to a novel110 kDa aminopeptidase in Heliothis virescens is not N-acetyl-galactosamine mediated. Insect Biochemistry and MolecularBiology 31: 909–918.

Burton SL, Ellar DJ, Li J, Derbyshire DJ, 1999. N-acetylgalactos-amine on the putative insect receptor amino peptidase N isrecognized by a site on the domain III lectin-like fold ofa Bacillus thuringiensis insecticidal toxin. Journal of MolecularBiology 287: 1011–1022.

Candy L, Peumans WJ, Menu-Bouaouiche L, Astoul CH, VanDamme J, Van Damme EJM, Erard M, Rouge P, 2001. TheGal/GalNAc specific lectin from the plant pathogenic basidio-mycete Rhizoctonia solani is a member of the ricin-B family.Biochemical and Biophysical Research Communications 282:655–661.

Carlini CR, Grossi-de-Sa MF, 2002. Plant toxic proteins withinsecticidal properties. A review on their potentialities asbioinsecticides. Toxicon 40: 1515–1539.

Coelho MB, Marangoni S, Macedo MLR, 2007. Insecticidal action ofAnnona coriacea lectin against the flour moth Anagastakuehniella and the rice moth Corcyra cephalonica (Lepidoptera:Pyralidae). Comparative Biochemistry and Physiology C 146:406–414.

Couty A, de la Vina G, Clark SJ, Kaiser L, Pham-Delegue MH,Poppy GM, 2001. Direct and indirect sublethal effects ofGalanthus nivalis agglutinin (GNA) on the development of

40 M. Hamshou et al.

a potato-aphid parasitoid, Aphelinus abdominalis (Hymenop-tera: Aphelinidae). Journal of Insect Physiology 47: 553–561.

Czapla TH, Lang BA, 1990. Effect of plant lectins on the larvaldevelopment of European corn borer (Lepidoptera: Pyralidae)and Southern corn rootworm (Coleoptera: Chrysomelidae).Journal of Economic Entomology 83: 2480–2485.

Davidowitz G, D’Amico LJ, Nijhout F, 2003. Critical weight indevelopment of insect body size. Evolution and Development 5:188–197.

Down RE, Gatehouse AMR, Hamilton WDO, Gatehouse JA, 1996.Snowdrop lectin inhibits development and fecundity of theglasshouse potato aphid (Aulacorthum solani) when adminis-tered in vitro and via transgenic plants, both in laboratory andglasshouse trials. Journal of Insect Physiology 42: 1035–1045.

Fitches E, Gatehouse AMR, Gatehouse JA, 1997. Effects of snow-drop lectin (GNA) delivered via artificial diet and transgenicplants on the development of tomato moth (Lacanobia oleracea)larvae in laboratory and glasshouse trials. Journal of InsectPhysiology 43: 727–739.

Gill SS, Cowles EA, Francis V, 1995. Identification, isolation, andcloning of a Bacillus thuringiensis CryIAc toxin-binding proteinfrom the midgut of the lepidopteran insect Heliothis virescens.Journal of Biological Chemistry 270: 27277–27282.

Hakim RS, Baldwin K, Smagghe G, 2010. Regulation of midgutgrowth, development, and metamorphosis. Annual Review ofEntomology 55, doi:10.1146/annurev-ento-112408-085450.

Hamshou M, Smagghe G, Van Damme EJM, 2007. Analysis oflectin concentrations in different Rhizoctonia solani strains.Communications in Agricultural and Applied Biological Sciences,Ghent University 72: 639–644.

Jaber K, Francis F, Paquereau L, Fournier D, Haubruge E, 2007.Effect of a fungal lectin from Xerocomus chrysenteron (XCL) onthe biological parameters of aphid. Communications inAgricultural and Applied Biological Sciences, Ghent University 72:629–638.

Kellens JTC, Peumans WJ, 1990. Developmental accumulation oflectins in Rhizoctonia solani: potential role as a storage protein.Journal of General Microbiology 136: 2489–2495.

Lemeire E, Borovsky D, Van Camp J, Smagghe G, 2008. Effect ofACE inhibitors and TMOF on growth, development and trypsinactivity of larval Spodoptera littoralis. Archives of Insect Biochem-istry and Physiology 69: 199–208.

Macedo MLR, Freire MGM, Silva MBR, Coelho LCBB, 2007. Insec-ticidal action of Bauhinia monandra leaf lectin (BmoLL) againstAnagasta kuehniella (Lepidoptera: Pyralidae), Zabrotes subfas-ciatus and Callosobruchus maculatus (Coleoptera: Bruchidae).Comparative Biochemistry and Physiology A 146: 486–498.

Maqbool SB, Riazuddin S, Loc NT, Gatehouse AMR, Gatehouse JA,Christou P, 2001. Expression of multiple insecticidal genesconfers broad resistance against a range of different rice pests.Molecular Breeding 7: 85–93.

Masson L, Lu YJ, Mazza A, Brosseau R, Adang MJ, 1995. TheCry1A(c) receptor purified from Manduca sexta displaysmultiple specificities. Journal of Biological Chemistry 270:20309–20315.

Mehlo L, Gahakwa D, Nghia PT, Loc NT, Capell T, Gatehouse J,Gatehouse A, Christou P, 2005. An alternative strategy forsustainable pest resistance in genetically enhanced crops.Proceedings of the National Academy of Sciences of the United Statesof America 102: 7812–7816.

Mier N, Canete S, Klaebe A, Chavant L, Fournier D, 1996. Insecti-cidal properties of mushroom and toadstool carpophores.Phytochemistry 41: 1293–1299.

Peumans WJ, Barre A, Hao Q, Rouge P, Van Damme EJM, 2000.Higher plants developed structurally different motifs torecognize foreign glycans. Trends in Glycoscience and Glycotech-nology 12: 83–101.

Peumans WJ, Van Damme EJM, 1995. Lectins as plant defenseproteins. Plant Physiology 109: 347–352.

Sadeghi A, Smagghe G, Jurado-Jacome E, Peumans WJ, VanDamme EJM, 2009. Laboratory study of the effects of leek lec-tin (APA) in transgenic tobacco plants on the development ofcotton leafworm Spodoptera littoralis (Lepidoptera: Noctuidae).European Journal of Entomology 106: 21–28.

Sadek MM, 2001. Polyandry in field-collected Spodoptera littoralismoths and laboratory assessment of the effects of male mat-ing history. Entomologia Experimentalis et Applicata 98: 165–172.

Sauvion N, Rahbe Y, Peumans WJ, Van Damme EJM,Gatehouse JA, Gatehouse AMR, 1996. Effects of GNA and othermannose binding lectins on development and fecundity of thepeach potato aphid Myzus persicae. Entomologia Experimentaliset Applicata 79: 285–293.

Shahidi-Noghabi S, Van Damme EJM, Smagghe G, 2008. Carbo-hydrate-binding activity of the type-2 ribosome-inactivatingprotein SNA-I from elderberry (Sambucus nigra) is a determin-ing factor for its insecticidal activity. Phytochemistry 69:2972–2978.

Shahidi-Noghabi S, Van Damme EJM, Smagghe G, 2009. Expres-sion of Sambucus nigra agglutinin (SNA-I0) from elderberry barkin transgenic tobacco plants results in enhanced resistance todifferent insect species. Transgenic Research 18: 249–259.

Shukla S, Arora R, Sharma HC, 2005. Biological activity ofsoybean trypsin inhibitor and plant lectins against cottonbollworm/legume pod borer, Helicoverpa armigera. PlantBiotechnology 22: 1–6.

Singh G, Rup PJ, Koul O, 2008. Selective feeding of Helicoverpaarmigera (Hubner) and Spodoptera litura (Fabricius) on meridicdiet with Bacillus thuringiensis toxins. Journal of Insect Behavior21: 407–421.

Smagghe G, Degheele D, 1994. Action of a novel nonsteroidalecdysteroid mimic, tebufenozide (RH-5992), on insects ofdifferent orders. Pesticide Science 42: 85–92.

Trigueros V, Lougarre A, Ali-Ahmed A, Rahbe Y, Guillot J,Chavant L, Fournier D, Paquereau L, 2003. Xerocomus chrysen-teron lectin: identification of a new pesticidal protein. Biochi-mica et Biophysica Acta 1621: 292–298.

Van Damme EJM, 2008. Plant lectins as part of the plant defensesystem against insects. In: Schaller A (ed), Induced PlantResistance to Herbivory. Springer, Dordrecht, The Netherlands,pp. 285–307.

Van Damme EJM, Lannoo N, Peumans WJ, 2008. Plant lectins.Advances in Botanical Research 48: 107–209.

Vandenborre G, Van Damme EJM, Smagghe G, 2009. Naturalproducts: plant lectins as important tools in controlling pestinsects. In: Ishaaya I, Horowitz AR (eds), Biorational Control ofArthropod Pests: Application and Resistance ManagementsSpringer-Verlag, Dordrecht, The Netherlands, pp. 163–187.

Vranken AM, Van Damme EJM, Allen AK, Peumans WJ, 1987.Purification and properties of an N-acetylgalactosamine spe-cific lectin from the plant pathogenic fungus Rhizoctonia solani.FEBS Letters 216: 67–72.

Wang H, Ng TB, Ooi VEC, 1998. Lectins from mushrooms.Mycological Research 102: 897–906.

Zhang HY, Liu XZ, Wei L, Zhou LY, Yang YM, 2007. Transgenictobacco plants containing Bt and GNA genes. BiologiaPlantarum 51: 746–748.