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S.K. Behura, Suresh Nair & Madan Mohan. ∗∗. International Centre for Genetic Engineering and Biotechnology (ICGEB), Aruna Asaf Ali Marg, New Delhi-110.
Plant Cell, Tissue and Organ Culture 64: 115–131, 2001. © 2001 Kluwer Academic Publishers. Printed in the Netherlands.

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Genetic, physiological and molecular interactions of rice and its major dipteran pest, gall midge Nagesh Sardesai∗ , K.R. Rajyashri∗ , S.K. Behura, Suresh Nair & Madan Mohan∗∗ International Centre for Genetic Engineering and Biotechnology (ICGEB), Aruna Asaf Ali Marg, New Delhi-110 067, India (∗∗ requests for offprints; Fax: +91-11-618-7680; E-mail: [email protected]) Received 17 February 2000; accepted in revised form 1 September 2000

Key words: biotypes, DNA fingerprinting, gall formation, gene pyramiding, insect resistance, map-based gene cloning, mapping and tagging, marker-assisted selection, Orseolia oryzae, Oryza sativa, resistance gene

Abstract The gall midge, Orseolia oryzae, is a major dipteran pest of rice affecting most rice growing regions in Asia, Southeast Asia and Africa. Chemical and other cultural methods for control of this pest are neither very effective nor environmentally safe. The gall midge problem is further compounded by the fact that there are many biotypes of this insect and new biotypes are continuously evolving. However, resistance to this pest is found in the rice germ plasm. Resistance is generally governed by single dominant genes and a number of non-allelic resistance genes that confer resistance to different biotypes have been identified. Genetic studies have revealed that there is a gene-for-gene interaction between the different biotypes of gall midge and the various resistance genes found in rice. This review discusses different aspects of the process of infestation by the rice gall midge and its interaction with its host. Identification of the gall midge biotypes by conventional methods is a long and tedious process. The review discusses the PCR-based molecular markers that have been developed recently to speed up the identification process. Similarly, molecular markers have been developed for two gall midge resistance genes in rice – Gm2 and Gm4t – and these markers are now being used for marker-assisted selection. The mapping, tagging and map-based gene cloning of one of these genes – Gm2 – has also been discussed.

Introduction Rice is the most important crop in the world with over 1.5 billion hectares under paddy cultivation and a worldwide production of over 596 million tons (FAO, 1999). It is grown in 117 countries, is a staple food in 39 countries and the major staple food for 2.7 billion people in Asia alone. Rice crop in the field is subjected to attack by a number of insect pests, pathogens, weeds and other harmful organisms. Several studies have reported that major yield losses of rice are often caused by insects alone. Cramer (1967) reported a yield loss due to insects in tropical rice of 34%. In India, annual yield loss has been calculated to vary from 28% (Kalode, 1987) to 35% (Way, 1976). Major rice insect pests that cause huge economic losses in Asia ∗ Both authors have contributed equally.

are the stemborer, rice bugs, leaf folder, leaf and plant hoppers, gall midge and rice hispa. The stemborer, brown plant hopper, gall midge and leaf hopper are among the most important insects in Southeast Asia and China, while in South Asia, the gall midge, brown plant hopper and yellow stem borer are the ones that cause major damage (Herdt and Riely, 1987). Of these insects, the gall midge alone causes a damage of more than US$700 million annually (Herdt, 1991). This review discusses various aspects of rice gall midge infestation, the primary symptom of which is the production of gall/s on the rice plant (Figure 1) and its interaction with its host. It also covers the various molecular studies that have been carried out so far in understanding this interaction along with molecular tools that have been developed to identify the gall midge resistance genes in rice as well as markers that easily identify the different biotypes of the insect.

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Figure 1. Susceptible rice variety TN1 showing galls. Lines indicate ‘onion shoot-like’ galls with an apical shortened lamina.

Two species of the rice gall midge have been identified so far, the Asian rice gall midge, Orseolia oryzae Wood-Mason (Figure 2A) and the African rice gall midge, O. oryzivora. Both species belong to the family Cecidomyiidae of the order Diptera. The Asian rice gall midge is a serious pest of rice in South and Southeast Asia (Ramasamy and Jatileksono, 1996). Severe losses due to this pest have been reported from Myanmar, China, Vietnam, Bangladesh, Sri Lanka, Indonesia, India and many other tropical Asian countries except Philippines and Malaysia (Reddy, 1967). In India, it is most prevalent in the states of Madhya Pradesh, Bihar, Orissa, Andhra Pradesh and Maharashtra (Chelliah et al., 1989). Recent reports have shown that it is also a major problem in Kerala and north eastern rice growing states in India. In contrast, the African rice gall midge, Orseolia oryzivora, is prevalent in Nigeria and many other parts of Africa

(Harris and Gagne, 1982; Ukwungwu et al., 1989). Although it is reported from many African countries, it has not attained the status of a major pest in most of them. The yield loss caused by gall midge is highly variable depending on the severity of attack. In extreme cases, complete yield loss of the crop has been observed. Studies indicate that for every unit percent increase in rice gall midge infestation, the yield loss is 0.502%, with losses varying among varieties and reaching as high as 2.5% in some of the varieties (Luh, 1982). In India, 25% yield loss per year over an area of 200,000 hectares is estimated (Lever, 1970).

Life cycle of the rice gall midge The life cycle from oviposition to adult emergence requires about two to three weeks (see review Joshi

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Figure 2. Stages in the life cycle of rice gall midge. (A) an adult rice gall midge; (B) a tip of the mature gall revealing the pupal case after the adult has flown from the gall; (C) a portion of the gall dissected out to show a pupa; (D) egg mass on leaf surface.

et al., 1983). Eggs are laid either on the hairs of the ligules of the rice leaf or on the leaf sheath (Figure 2D) or on the underside of the leaf blade (Sontakay, 1941), either singly (Yen et al., 1941) or in groups of two to six (Sontakay, 1941). The egg-laying capacity of female gall midges varies greatly and could be anywhere from 100 to 400 eggs, an average being 200 (Yen et al., 1941; Wong et al., 1956). The incubation period of the eggs varies from three to four days and on hatching, the tiny young larvae creep down between the leaf sheaths until they reach the growing point of the apical or lateral buds. High relative humidity, enough to cause condensation on the leaf surface, is essential for larval movement. Young larvae, upon hatching are about 1 mm long, pale and distinctly divided into 13 segments. Larval period in rice gall midge varies from 6–33 days (Sontakay, 1941; Huang, 1957; Fernando, 1962). On entering the interior of the bud the larvae lacerate plant cells and feed. The presence of an active first instar larvae at the meristem stimulates the formation of a gall and suppresses the development of the growth cone. The larvae continue to feed at the base of the growth cone during instars 2 and 3. When fully fed, third instar larvae molt into pupae (Figure 2C). By the time pupation takes place the gall cavity reaches a length of about 190 mm. The fully formed gall (Figure 2B) consists of a long ivory-white tube that terminates in a solid plug of white tissue.

Above this plug the rest of the leaf sheath is delimited from a shortened lamina by a distinct ligule. The pupal period in rice varies from two to eight days (Murthy, 1958). As adult emergence stage approaches, the pupa wriggles its way up the tube to the tip of the silver shoot and when ready to transform into a midge, bores a hole at the upper end of the gall cavity and projects itself halfway out. The skin of the pupa then bursts and the midge crawls out, dries its wings, hangs on to the pupal skin and flies away (see review Joshi et al., 1983). Females have been observed to have a life span of 1–5 days while males live for 1–2 days (Li and Chiu, 1951). Copulation takes place soon after emergence and within a few hours females start laying eggs (Sontakay, 1941). Females copulate only once whereas the males can mate multiple times (Wong et al., 1956). Five to seven overlapping generations in a year have been reported in rice (Yen et al., 1941; Li and Chiu, 1951; Wong et al., 1956). The rice gall midge produces monogenous progeny wherein all offspring of a single female are of one sex only (Sain and Kalode, 1988). An interesting feature of the gall midge life cycle is the existence of a female biased sex ratio in the population with the ratio of male-producing females and female-producing females being 1:1.32 (Panda and Mohanty, 1970).

118 Ecological and cultural factors influencing gall midge infestation It has been observed that rains during the daytime and high humidity favour insect infestation (Wong et al., 1956). Though high humidity is necessary to breed the insect, fields with high and low water levels or with good or bad drainage do not show any difference in incidence (Patel et al., 1957; Pai and Rao, 1958). It has also been observed (Murthy, 1958) that infestation increases with the lateness of sowing the first crop during the monsoon. A comprehensive study made by Descamps (1956) reveals that after harvest of the rice crop, the pest infests wild rice, Oryza barthii, which grows abundantly in and around water. As it dries up in summer, most of the larvae enter diapause in the buds of the plants. Under favorable moisture conditions, development may continue throughout, otherwise, the diapause continues for many months. Wong et al. (1956) have reported that larvae overwinter in the stubble from November to April and then pupate.

Methods of controlling gall midge populations Although different methods of control have been suggested, no definite recommendations are available. Mechanical, cultural and chemical measures and the use of resistant varieties have been recommended. Mechanical and cultural control Weeding out of infested plants and of alternate hosts to keep insect populations down followed by immediate plowing of stubble after harvest have been recommended as control measures (Wong et al., 1956; Israel, 1959; Krishnamurthy Rao and Krishnamurty, 1960). Early sowing and transplanting to avoid gall midge attack has been practiced successfully (Hegdekatti, 1927). Biological control More than 95% of gall midge larvae are parasitized by Platygaster sp. (order Hymenoptera, suborder Apocrita, group Parasitica) late in the rice growing season (Kobayashi et al., 1991). Introduction of parasites of gall midge into new areas where they do not exist has been suggested (Sontakay, 1941) as another method of control. Other chalcids and braconids that parasitize the larvae and keep them in check have also been

reported (Ladell, 1933; Sontakay, 1941; Li and Chiu, 1951). Though detailed studies confirm that biological control is a very effective means of checking the pest (Descamps, 1956), parasitization of gall midge eggs by Platygaster does not prevent the former from hatching into larvae and forming galls on the plant. Thus, parasitization does not prevent one round of infestation by the gall midge though a high incidence of parasitization may have a check on the further increase of gall midge infestation in a growing season. Besides, Platygaster usually appears quite late in the life cycle of the rice gall midge to have a major effect in checking the midge population. Chemical control Work on the use of chemicals for the control of the rice gall midge was started only in the early 1950s and effective control of the pest was obtained by spraying the crop with BHC during the growing season (Wong et al., 1956). Other insecticides such as dieldrin, endrin, ekatin, parathion, diazinon, malathion and cupravit have also been employed to control the midge (Patel and Bhat, 1954; Israel et al., 1959; Ou and Kanjanasoon, 1961). However, the use of these chemical control measures is neither completely effective nor environmentally safe. Thus, the only effective and safe way to control gall midge infestation is through the deployment of gall midge resistance genes.

Biotypes of gall midge One of the major problems faced by the rice breeders is the emergence of new gall midge biotypes. Midge biotypes are morphologically indistinguishable and capable of interbreeding but differ in their reaction to genetically defined rice varieties (Grover and Prasad, 1980). The first possible evidence on the occurrence of biotypes in gall midge was presented by Khan and Murthy (1955) who reported that the variety HR14 was consistently less susceptible than HR8 at Nizamabad (India). Differential reactions of test donors in different countries has also been reported (Fernando, 1962). Currently, a total of 13 biotypes of the Asian rice gall midge are reported in literature (Table 1). Indian biotypes have been identified and characterized on the basis of differential reactions to genetically defined host plants. The results of the differential screenings are outlined in Table 2.

119 Table 1. The number of rice gall midge biotypes reported from different countries

volving gall midges collected from Thailand and India it was found that the abdomens of midges collected in Thailand were covered with longer and softer hairs than the gall midges collected in India. This indicated that differentiation has occurred between the populations of Thailand and India. However, no distinct morphological variations have been observed between the biotypes of the gall midges identified in India.

Country

Biotypes reported

References

India

5

Kalode and Bentur, 1989 Nair and Devi, 1994 Anonymous, 1992

China

4

Lai et al., 1984 Tan et al., 1993

Sri Lanka

2

Anonymous, 1981

Karyological variations

Thailand

1

Anonymous, 1981

Indonesia

1

Anonymous, 1981

Studies have been carried out in rice gall midge to get an insight into its mitotic and meiotic chromosome behavior and mechanisms of sex differentiation. However, these studies have not been able to provide a tool for biotype differentiation. Investigations of the mitotic chromosomal preparations from cerebral ganglia of biotype 2 of gall midge have revealed a karyological sexual dimorphism – with females having 8 chromosomes and males having only 6 chromosomes in the somatic cells (Sahu et al., 1996). The female soma has two pairs of autosomes and two pairs of sex chromosomes; the male somatic cell has 2 pairs of autosomes, but only one each of the two sets of sex chromosomes – these sex chromosomes are maternally derived. The paternal set of the sex chromosomes is eliminated during early embryogenesis in the males (Sahu et al., 1998). Similar sexual dimorphisms have been observed in many species belonging to the Cecidomyiidae (White, 1950; Stuart and Hatchett, 1988).

There are examples of the existence of biotypes among other insect pests as well (see review Smith, 1998). The Cecidomyiid, Hessian fly- Mayetiola destructor is one of the well-studied agricultural pests of wheat for existence of biotypes for which at least 16 biotypes were identified (Sosa, 1981; Ratcliffe et al., 1994). Another example of a well-studied pest is the brown plant hopper (BPH), Nilaparvata lugens, a pest of rice. Varieties of rice differ greatly in their susceptibility to plant hopper. Four biotypes of BPH (Verma et al., 1979) and four major resistance genes have been identified (Sogawa, 1982). The process of identification of different biotypes solely based on host plant differential reactions and on the basis of midge feeding behaviour has slowed biotype identification resulting in the delay in deployment of resistant varieties of rice in gall midge prevalent areas. This has also slowed down the process of breeding new rice varieties that are resistant to gall midge. Moreover, host differentials cannot be exploited using a single gall midge representing a biotype for which a genetically related group of insects is a requisite for biotype identification. Therefore, alternate methodologies using morphological, karyological, enzyme or DNA-based differences to identify biotypes would be an improvement over conventional methods of identification. Morphological variations Environmental factors have been reported to affect different traits of morphology and behavior in insects (Diehl and Bush, 1984). In a comparative study in-

DNA fingerprinting In recent years, DNA-based molecular markers have been extensively used in many areas such as gene mapping and tagging, genome identification, analysis of genetic diversity and estimation of genetic relatedness (Karp and Edwards, 1997; Mohan et al., 1997a; Katiyar et al., 2000). DNA-based methodologies have been found to be the best alternatives to differentiate closely related organisms (Goodwin and Annis, 1991; Mohan et al., 1997b). The use of DNA-based markers allows efficient comparison because genetic differences are detectable at all stages of development of the organism unlike isozymes which may show age dependent changes (Loxdale et al., 1996). DNA-based differences are also unaffected by environmental factors as compared to morphological and isozyme markers

120 and have better resolving capability in revealing the genetic differences (Wilson, 1976). Extensive work has been done in revealing the molecular differences in insect strains through RFLP of mitochondrial DNA at the interspecific levels (Shah and Langley, 1979; Trick and Dover, 1984). Ribosomal RNA genes have been widely used in many insect species to understand the genetic variability and for inferring the evolutionary relationships amongst them (Barciszewska et al., 1995; Campbell et al., 1995; Pawlowski et al., 1996). Mitochondrial ribosomal RNA genes have also been used for molecular identification of sibling species by PCR-based methods (Tang et al., 1995). Using the Orseolia oryzivora 12S rRNA fragment as RFLP probe, it was possible to distinguish between the Asian and African gall midges (Behura, 1999). However, this probe could not differentiate the biotypes of Asian gall midge. Recently, DNA-based molecular markers have been developed using random amplified polymorphic DNAs (RAPDs) that can be used for biotyping of the Asian rice gall midge and sequence characterized amplified region (SCAR) markers have been developed to identify different species and biotypes of gall midge in a multiplex PCR-based assay (Behura et al., 1999). However, the multiplex PCR-based assay, could differentiate biotype 2 only by default as no product was amplified using these primer sets. To overcome this problem, amplified fragment length polymorphism (AFLP) technique has been successfully exploited to differentiate biotype 2 from the other biotypes. Although the AFLP marker is shared by two other biotypes, biotype 1 and biotype 5, it differentiates biotype 2 from the other biotypes when used in a multiplex PCR with the SCAR primers derived from RAPDs (Behura et al., 2000). DNA-based molecular markers have been used earlier for differentiating between biotypes of the white fly, Bemisia tabaci (Gawel and Bartlett, 1993). Similarly, as far back as 1976, Gorske and Sell assessed the genetic differences between two biotypes of purslane sawfly (Schizocerella pilicornis), to clarify their biotype status in the population.

Response of rice plant to gall midge infestation The infestation of rice by gall midge results either in, (A) induction of gall formation by the larvae in susceptible varieties; or (B) responses to infestation in resistant varieties.

Figure 3. A longitudinal section through a 8-day-old gall showing the growing insect larva, the host’s apical meristem (growth cone) and the fully developed apical plug in the gall.

A) Induction of gall formation in susceptible varieties Although the factors influencing gall midge infestation have been well studied, those inducing the formation of galls in rice are not known. When larvae reach the apical meristem and start feeding at the base of the growth cone, the cells of the innermost leaf primordium, are induced to proliferate. These cells grow in a radial fashion and finally fuse to form a plug, enclosing the larva within it (Figure 3). The presence of an active larva at the growing meristem triggers gall formation and is a prerequisite not only for induction, but also for the continued development of the gall. Rendering the gall midge larva inactive by the use of insecticides affects gall formation – the effect on the gall depends on the stage at which the larva is killed or rendered inactive. If the larvae are at the first or early second instar stages, before the growing tissue has fused to form a plug, death or inactivity of

121 the larva results in elongation of the gall and renewed activity of the growth cone within three days (Perera and Fernando, 1968). However, if the larvae were at the late second instar stage, the leaf primordia differentiates and grows as at the earlier stages, but once the growing leaf reaches the upper limit of the gall cavity, it becomes wrinkled due to lack of space. Killing the third instar larva or pupa results in greater elongation of the gall, but the growth cone is not reactivated and the plant grows further only by tillering. It has been suggested that the induction of cell proliferation and hence gall formation, is through production of a molecular ‘signal’ – a ‘cecidogen’ from the salivary gland of the feeding larvae (Chiu, 1980). However, it has not yet been established whether it is the salivary gland substance released by the larva, an excretory product released from the posterior end of the larvae or the feeding activity of the larva that induces the growth of the gall. Factors affecting gall midge resistance in rice Physical and chemical factors A number of factors had earlier been implicated in providing the rice plant with resistance to gall midge. Morphological characters like purple colour, scent possessed by the plant (Anonymous, 1952) or hairiness of the leaves (Krishnamurthy Rao and Krishnamurthy, 1964) were implicated as factors involved in providing resistance to a particular biotype. Rao et al. (1971) suggested that the compactness of leaf sheath prevents the entry of the maggot in some varieties. However, it was later confirmed that such physical barriers have little relation to the ability or inability of the insect to infest the rice plant (Shastry et al., 1972). Similarly, certain biochemical factors were also implicated in conferring resistance to gall midge. The presence of high amounts of phenolic compounds and amino acids have been reported to contribute to the plant resistance (Vidyachandra et al., 1981). However, analysis of either the basal stem portion or the whole plant sample of twenty-nine gall midge resistant and susceptible varieties for total phenol content did not reveal any correlation with resistance (Amudhan et al., 1999). Thus, a clear association has not been shown so far to indicate that phenols play a role in resistance. Genetic factors The study of inheritance of resistance in many different rice varieties led to the conclusion that resistance

to gall midge is, in most cases, a monogenic character controlled by a single dominant gene (Satyanarayaniah and Reddi, 1972; Chaudhary et al., 1986; Tomar and Prasad, 1992). Chaudhary et al. (1986) investigated the relationships of the resistance genes in different varieties by crossing five different resistant cultivars – Usha, Samridhi, Bd6-1, Surekha and IET6286. While all five varieties were found to have a single dominant gene for resistance, allelic tests revealed that three of the varieties – Usha, Samridhi and Bd6-1 had the same gene for resistance – the gene was designated Gm1; while the other two varieties had another dominant gene non-allelic to Gm1 and was hence designated Gm2. Other non-allelic gall midge resistance genes have been identified subsequently. They include gm3 from RP2068-18-3-5 (Kumar et al., 1999), Gm4(t) from Ptb 10 (Shrivastava et al., 1994), Gm5 from ARC5984 (Kumar et al., 1999), Gm6 from the Chinese cultivar Duokang #1 (Yang et al., 1997) and Gm7 from ARC 10659 (Kumar et al., 2000). All these genes, except for gm3, are dominant resistance genes (Table 3). When standard differentials (varieties used for biotyping) were exposed to the different gall midge biotypes, it was seen that many of the rice differentials showed resistance to more than one biotype. While TN1 was susceptible to all the biotypes, studies on W1263 (Sastry et al., 1984) which derives its resistance from Eswarakora, indicates that a single dominant gene confers resistance – this gene has been designated Gm1. Exposure of W1263 to different biotypes of gall midge reveal that W1263 shows resistance to the biotypes 1, 3 and 5. As the resistance in W1263 is conferred by a single gene, it would indicate that Gm1 confers resistance to biotypes 1, 3 and 5. Similarly, Phalguna, carrying the dominant resistance gene Gm2 shows resistance to the biotypes 1, 2 and 5 (Pasalu and Rajamani, 1996) while RP2068-183-5 with the recessive gene gm3 shows resistance to biotypes 1, 2 and 3 (Kumar et al., 1999). The Gm4 gene provides resistance to biotypes 1, 2, 3 and 4 and Gm5 to biotypes 1, 2 and 5 (Pasalu and Rajamani, 1996). The Gm6 gene from the cultivar Duokang #1 confers resistance to all four biotypes of gall midge identified from China (Tan et al., 1993). Thus, the different gall midge resistance genes provide resistance against different sets of biotypes of the insect (Bentur and Amudhan, 1996). A table of the gall midge resistance genes and the biotypes they provide resistance against is given (Table 4).

122 Table 2. Biotype identification based on host differential reactions Biotype

1 2 3 4 5

Eswarakora

Hosts Siam29

Velluthacheera

References

R S R S R

R R S S R

R R R R S

Kalode and Bentur, 1989 Kalode and Bentur, 1989 Kalode and Bentur, 1989 Bentur et al., 1987 Pasalu and Rajamani, 1996

R: Resistant; S: Susceptible. Table 3. Rice gall midge resistance genes, their sources and donors Resistance gene

Source

Donor

Reference

Gm1 Gm2 gm3 Gm4 Gm5 Gm6 Gm7

Eswarakora Siam29 Velluthacheera Ptb10 ARC5984 Duokang #1 ARC10659

Samridhi, Ruchi, Asha Surekha, Phalguna RP2068-18-3-5 Abhaya, R296-260 ARC5984 Duokang #1 RP2333-156-8

Chaudhary et al., 1986 Chaudhary et al., 1986 Kumar et al., 1999 Shrivastava et al., 1994 Kumar et al., 1999 Yang et al., 1997 Kumar et al., 2000

B) Responses to infestation in a resistant variety Rice varieties showing resistance to gall midge respond to infestation in two ways: – Some resistant varieties show a hypersensitive response (HR) wherein the host cells around the larva undergo apoptotic cell death. Dead larvae are observed within the plug of dead tissue. Extensive necrosis of the meristem accompanied by varied degrees of central leaf browning and at times, death of the entire leaf is observed. HR is accompanied by the induction of a general defense mechanism known as systemic acquired resistance. – Other resistant varieties do not exhibit HR, although they do not allow the larvae to grow. It is not known whether resistance in these varieties is due to the production of certain defense chemicals or due to lack of nutritive elements necessary for the larvae to survive. Bentur and Kalode (1996) screened over 130 gall midge biotype 1 – resistant rice varieties using biotype 1 of the insect and found that while some varieties express HR, others do not, although they all remain resistant to this biotype.

Phalguna is a variety that shows resistance to the gall midge biotypes 1, 2 and 5, while it is susceptible to biotypes 3 and 4. Bentur and Kalode (1996) observed that Phalguna plants respond to infestation by gall midge biotype 1 (GMB1) with a hypersensitive response. If the secondary tillers were infested with the same biotype within 21 days of the infestation of the primary tillers, no HR was observed in the latter. However, if there was a time lag of 28 days or more between the primary and secondary infestations, HR was observed in the secondary tillers 5 days after the secondary infestation. All the larvae are killed within 5 days regardless of the time interval between the two infestations. This suggests that the HR is not essential for larval mortality. It was also observed (Bentur and Kalode, 1996) that when Phalguna seedlings were infested with GMB1 followed by a second round of infestation with GMB4, a HR was observed and the GMB4 larvae were killed in 70% or more of the plants. The resistance induced against GMB4 was effective for 4 weeks. Similarly, infestation with GMB4 followed with GMB1 infestation within 4–6 days also led to a HR and larval mortality in most of the plants. Thus, the HR in Phalguna is induced by the avirulent biotype 1 irrespective of whether infestation by this biotype

123 Table 4. Differential reaction of different gall midge biotypes to known rice resistance genes Gene

Gm1 Gm2 gm3 Gm4 Gm5 Gm6 Gm7

1

2

R R R R R S R

S R R R R – R

Biotype 3 4 R S R R S – S

S S S R S – R

5 R R S S R – –

R: Resistant; S: Susceptible; – not tested; Note: References related to the screening of the respective resistance genes are as given in Table 1.

is primary or secondary to infestation by some other biotype. However, secondary infestation with GMB1 eight days after GMB4 infestation resulted in mortality of the GMB1 larvae in only 35% of the plants. In the remaining plants, HR was localized and the GMB1 larvae in secondary tillers died, but the GMB4 larvae survived in the primary tillers. Whether exposure to the pest results in a HR+ or HR− reaction appears to depend upon the resistance gene the variety carries. It has been observed that varieties with the Gm2 gene show HR+ response, while those with Gm1 show HR− response. Results of crosses between varieties carrying Gm2 with those carrying Gm1 showed that HR+ response is epistatic to HR− response (Bentur et al., 1998). However, the nature of the signals/molecules involved in this kind of protection/resistance is not known.

Gene-for-gene hypothesis as a means to understand gall midge-rice interaction Flor (1971) suggested a gene-for-gene hypothesis to explain disease resistance in plants. According to this hypothesis, the product of a pathogen avirulence (avr) gene, or a factor produced by it, is recognized by a corresponding plant disease resistance (R) gene product and thereby triggers the resistance response. Subsequent, studies have justified this hypothesis (see review Staskawicz et al., 1995). In the case of gall midge interaction with rice it has been observed that exposure to the pest triggers two

types of plant resistance responses – a general nonhost resistance response and a specific race/cultivarspecific host resistance response. Non-host resistance is exhibited by all plant species that respond to potential pathogens without apparent R/avr gene combinations. The race/cultivar-specific host resistance response involves the recognition of the gene product of an avirulence (avr) gene specific to the particular pathogen by the product of the resistance (R) gene of the host. This probably involves two steps: – recognition of the avr gene product by the R gene product; – triggering of downstream genes leading to the resistance. It is postulated that the avr gene of the pathogen encodes a protein molecule that is recognized by a matching receptor in a particular plant genotype. The formation of the receptor-protein complex initiates the hypersensitive response and other plant defense responses (see review De Wit, 1997). Various Arabidopsis mutants compromised in disease resistance have been identified and these mutants have been used to dissect the different components of the disease resistance pathway (see review Gopalan and He, 1998). Such gene-for-gene interactions as those mentioned above have been well documented in the interaction of the biotypes of greenbug and sorghum (Puterka and Peters, 1995), raspberry aphid Amphorophora udaci with its host (Briggs, 1965) and the Hessian fly, Mayetiola destructor and its wheat host (Hatchett and Gallun, 1970; Gallun, 1978). In wheat, 27 different genes that confer resistance to the Hessian fly have been described (Ratcliffe and Hatchett, 1997), while a number of different biotypes of the Hessian fly have also been identified. The Hessian fly resistance genes are dominant over their susceptible alleles, while single genes in the insect confer avirulence to specific resistance genes in the host (Gallun and Hatchett, 1969). Studies involving six different resistant genes in wheat, H3 (Gallun and Hatchett, 1969), H7, H8 (Hatchett and Gallun, 1970; Patterson and Gallun, 1977); H6 (Gallun, 1978), H5 (Perry, 1990), H9 (Formusoh et al., 1996) and H13 (Zantoko and Shukle, 1997) show that each of these resistance genes has a corresponding avirulence gene in the Hessian fly. The interaction between rice and gall midge shows great similarity to that between Hessian fly and wheat. At least six non-allelic gall midge resistance genes (in rice) and 5 different biotypes of the gall midge have

124 been identified in India. Studies by Bentur et al. (1992) have suggested that, as in the case of the Hessian fly, the gall midge also may have a gene-for-gene interaction with its host. Resistance in rice is monogenic and is conferred in most cases by dominant genes, while the virulence in gall midge is probably controlled by recessive virulence genes. Bentur et al. (1998) observed that virulence of gall midge biotype 4 against Phalguna, containing the Gm2 gene, is inherited as a single recessive gene. Using host plant differentials, Behura et al. (2000) observed that the female progeny of a cross between a female parent virulent to Phalguna (biotype 4) and a male avirulent to Phalguna (biotype 1 and 2), are all avirulent, whereas the male progeny of this cross are virulent. Since it has already been observed that the male midges are monosomic for the sex chromosomes that are also maternally derived, the above results suggest that the avirulence gene in biotype 4 is X-linked. The female progeny being diploid for the X chromosomes are all avirulent (since avirulence is dominant over virulence), while the male progeny that inherit only the maternally derived X chromosomes are virulent when the mother is of a virulent biotype and avirulent when their mother is avirulent. Behura et al. (2000) also identified an AFLP marker that segregates along with the avirulence gene. Three of the avirulence genes – vH6, vH9 and vH13 that confer avirulence to Hessian fly resistance genes H6,H9, and H13 in wheat in a gene-for-gene relationship have recently been shown to be X-linked (Stuart et al., 1998; Schulte et al., 1999).

Mapping and tagging of gall midge resistance genes Attempts are being made to map, tag and clone the gall midge resistance genes in rice. The first gall midge resistance gene to be mapped is the Gm2 gene that confers resistance to biotypes 1, 2 and 5 of gall midge (Mohan et al., 1994). RFLP mapping was performed on a mapping population of 40 recombinant inbred (RI) lines (F5−6 ) derived from a cross between Phalguna, a resistant variety and ARC 6650, a susceptible variety, using 150 RFLP probes. The results revealed that Gm2 segregated closely with four RFLP markers that map to chromosome 4 of rice. RAPD (random amplified polymorphic DNA) analysis of the same population using 520 RAPD primers allowed the identification of two RAPD fragments which are

tightly linked to the Gm2 gene – a 1.7 kb fragment (F8) present in all susceptible RI lines and a 0.6 kb fragment (F10) present in all resistant RI lines (Nair et al., 1995). F8 and F10 have also been mapped and are found to be at 4.1 and 5.4 cm distance from Gm2 respectively (Mohan et al., 1994). F8 and F10 are now being used as molecular tags to study the inheritance of the Gm2 gene. Similarly, the gall midge resistance gene Gm4t has also been tagged and mapped (Nair et al., 1996; Mohan et al., 1997b). Rice DNAs from the gall midge resistant variety Abhaya which carries the Gm4(t) gene and a susceptible variety Tulsi and their F3 progeny were screened using 500 random primers in order to detect a random amplified polymorphic DNAs (RAPDs) linked to the gene, Gm4(t). One of these primers, E20, amplified two bands of 570 bp and 583 bp, which were tightly linked to resistance and susceptibility, respectively. These bands were cloned and sequenced and primers were designed such that they amplify the 583 bp fragment specifically in susceptible lines and the 570 bp fragment in resistant lines and can now be used for marker aided selection of the Gm4(t) gene in rice. Attempts were also made to map the 570 bp resistance specific marker in a cross between two indica parents, Abhaya (resistant) × Shyamala (susceptible). However, no useful polymorphism could be detected between the parents and thus this marker could not be mapped. Subsequently, E20570 was mapped using another mapping population derived from a cross between Nipponbare, a japonica variety and Kasalath, an indica variety, in which Gm4t is not known to be present. Using the population from this cross, E20570and hence Gm4(t) which is tightly linked to it, could be mapped to chromosome 8 between markers R1813 and S1633B (Mohan et al., 1997b). This also represents an alternate strategy for mapping resistance genes using a population not harboring the gene. Some of the other insect resistance genes which have been mapped using RFLP and RAPD techniques are hessian fly resistance genes in wheat (Dweikat et al., 1994, 1997; Gill et al., 1987; Ma et al., 1993), brown plant hopper resistance genes in rice (Ishii et al., 1994) and aphid resistance gene in cowpea (Myers et al., 1996).

Marker Assisted Selection (MAS) for gall midge resistance genes In breeding for disease and pest resistance, the se-

125 gregating populations derived from crosses between the resistant sources and plants with desirable agronomic traits are selected either at natural or artificially created disease and pest nurseries with high insect pressure, or by infecting individual plants under controlled environments. In the case of the gall midge, screening is based on the natural occurrence of the pest that is limited to one particular time of the year, i.e. just 2–4 months following monsoon. Moreover, in India, different biotypes of gall midge are distributed in different regions of the country and normally two or more biotypes do not occur together at the same geographical location. Consequently, the selection for rice plants resistant to more than one biotype of gall midge becomes very time consuming. For example, if plants resistant to biotype 1 have to be screened for resistance to another biotype, they will have to be screened at the second site where the second biotype is prevalent. As the time of the annual appearance of all biotypes in India is more or less same, screening for the second biotype will have to be postponed till the following year. This makes the process of breeding and pyramiding of gall midge resistance genes a labor intensive and time consuming process. The development of molecular markers that are tightly linked to the gene of interest would enable one to follow the gene in a cross intended to breed new resistant varieties any time of the year without depending on the annual occurrence of insects. Many rounds of selection can be carried out in a year even in the absence of the pest (see review Mohan et al., 1997a). In order to make marker assisted selection for the gall midge resistance genes possible, markers have been developed which show close linkage to the resistance genes. Nair et al. (1995) have identified an allelespecific PCR-based marker linked to the Gm2 gene in rice. Two RAPD fragments were found to be tightly linked to the Gm2 gene – a 1.7 kb fragment present in all susceptible RI lines and a 0.6 kb fragment present in all resistant RI lines. These fragments were cloned and partially sequenced and the sequence information used to design 24-mer PCR primers. When the two sets of primers were used in a PCR with the parental DNAs as template, a 1.7 kb fragment amplified from the susceptible parent and failed to amplify from the Phalguna genome. In contrast, the primer specific to the 0.6 kb fragment amplified a 0.6 kb fragment from Phalguna and failed to amplify at all when the susceptible parent was the template. When these primer sets were used together in a single PCR (multiplex) to amplify DNA from the parents and the RI lines derived

from the Phalguna × ARC6650 cross, the amplification pattern revealed that the 1.7 kb fragment alone was amplified in the susceptible parent and the susceptible progeny lines and the 0.6 kb fragment alone in the resistant parent and the resistant lines. These markers are now being used for marker assisted selection for the Gm2 gene in breeding programmes (Figure 4A). Similarly, the two RAPD fragments amplified with the E20 primers which are tightly linked to Gm4(t) gene (see mapping and tagging section) have been cloned and sequenced. The primers designed from the sequence amplify a 583 bp fragment in the susceptible parent and all the susceptible F3 individuals derived from a cross between Abhaya (resistant) and Tulsi (susceptible), whereas, the resistant parent and the resistant F3 progeny amplify the 570 bp fragment (Nair et al., 1996). These primers are also being used in breeding programmes (Figure 4B).

Cloning of the gall midge resistance gene, Gm2 Although two of the gall midge resistance genes have been mapped and tagged, none of them have been isolated or cloned. In fact, no insect resistance genes have been cloned from rice so far. However, attempts are being made to clone some of these genes by different methods including map based cloning and homology based cloning. Our lab has taken a map-based as well as homology based approach to clone the gall midge resistance gene Gm2.

Map based cloning approach As mentioned earlier, the Gm2 gene is linked to and flanked by five RFLP markers – RG214, RG329 on one side and RG476, F8 and F10 on the other (Mohan et al., 1994). These five markers have been used to screen a YAC (Yeast artificial chromosome) library constructed from the Nipponbare genome. 20 YAC clones were found to hybridize to these markers, ten of which were overlapping clones forming a contiguous stretch of DNA in this region (Yoshimura et al., 1996; Rajyashri et al., 1998). On the basis of Southern hybridization of these markers to the ten YAC clones, a physical map of this region has been derived. All the markers flanking the Gm2 gene hybridize to two YACs – Y2165 (300 kb) and Y3487 (500 kb). Of these, YAC Y2165 (300 kb long) hybridizes to the markers RG214

126

Figure 4. A schematic representation of PCR-based marker aided selection (MAS) for gall midge resistant (R) and susceptible (S) lines in rice using multiplex allele specific PCR. P1 and P2 are the susceptible and resistant parents, respectively. The gall midge resistance genes being selected for are Gm2 (A) and Gm4t (B). The figures on the left represent molecular weights. Progenies are individuals arising out of a cross between the parents P1 and P2. In the case of MAS for Gm2 the progeny used is a F5 -F7 population and in the case of Gm4t it is a F3 population (For details see Nair et al., 1995, 1996). By following the inheritance pattern of the bands it is possible to predict the resistance/susceptible nature of the progeny. Individuals of the progeny inheriting the 1.7 kb or the 583 bp fragment are susceptible and those inheriting the 0.6 kb and 570 bp fragments are resistant. Individuals amplifying both bands are resistant as both Gm2 and Gm4t are dominant genes.

and RG329 which flank Gm2 on one side, as well as to F8 which flanks it on the other. Thus, the allele for the Gm2 gene has been localized to within 300 kb in the Nipponbare genome. However, Nipponbare does not carry the Gm2 gene. Hence a cosmid library of the region covered by Y2165 has been constructed from the Phalguna genome carrying the Gm2 gene and at-

tempts are on to clone the Gm2 gene present within this contig (Figure 5). Homology based cloning approach Genes conferring resistance to many different classes of pathogens including bacteria, viruses, fungi and

127

Figure 5. An outline of a strategy being used to isolate the gall midge resistance gene, Gm2, from Phalguna, an indica rice variety. YAC: Yeast artificial chromosome. (For details see Rajyashri et al., 1998).

nematodes have been isolated from different plant species. Sequence analysis of the predicted proteins of the cloned disease resistance genes reveals that most resistance genes of diverse origin and pathogen specificity possess certain conserved amino acid motifs. These motifs consist of nucleotide-binding-site (NBS) domain and a hydrophobic domain (HD) with a consensus amino acid sequence Gly-Leu-Pro-Leu (GLPL) downstream of the NBS. Using degenerate primers for amplifying these conserved regions, many investigators have amplified homologous regions from the genomes of diverse plant species (Kanazin et al., 1996; Leister et al., 1996, 1998; Yu et al., 1996; Feuillet et al., 1997; Aarts et al., 1998; Collins et al., 1998; Shen et al., 1998; Speulman et al., 1998; Mago et al., 1999). The first resistance gene to be identified that affects insects is the Mi gene that confers resistance to the root

knot nematode in tomato which when introduced into potato confers resistance to the aphid Macrosiphum euphorbiae. This gene, like other disease resistance genes, also codes for a protein that contains the NBS and LRR regions (Milligan et al., 1998; Rossi et al., 1998). Using primers that amplify the conserved NBSLRR sequences, it was thought possible to amplify other insect resistance genes like the gall midge resistance gene. Taking this homology based approach, Mago et al. (1999) have PCR-amplified resistance gene analogues (RGAs) from rice in order to identify clones that segregate with the gall midge resistance genes. Several of the RGAs isolated in this study were found to be linked to known resistance genes. Mago et al. (1999) were interested in seeing if these RGAs segregate with the gall midge resistance genes. In fact, one of the RGAs – RGA7, which mapped to chromosome 4, showed cosegregation with markers G235 and G264 and close linkage to V17. These markers are also known to be tightly linked to Gm2 (Rajyashri et al., 1998) and Xa1 gene (Yoshimura et al., 1996). RGA 7 also hybridized to the same YAC as the Gm2 flanking markers upon hybridization with the Nipponbare YAC library. It is possible that RGA 7 is a portion of Gm2 gene which we are in the process of verifying. It is known that resistance genes often occur in clusters at a single locus (Kanazin et al., 1996; Leister et al., 1996, 1998; Yu et al., 1996). The Xa21 locus is a cluster of eight genes, including the Xa21 gene that confers resistance to bacterial blight – all of which show a very high degree of sequence homology to each other – ranging from 63.5%–98% identity. Seven of the eight members of this cluster share a highly conserved 233 bp sequence (Song et al., 1995, 1997). It has been proposed that duplication, recombination and transposition contribute to the amplification and diversification of the Xa21 gene family. The mapping of Gm2 (Mohan et al., 1994) as well as Xa1 (Yoshimura et al., 1996, 1998) to a region very close to each other suggests that these resistance genes may also occur in a similar cluster. Perhaps more detailed examination of the region around the gall midge resistance gene would reveal the presence of clusters of similar genes which differ from each other only in the biotype to which they are specific. Thus, different approaches are being taken, at present, to clone the gall midge resistance genes. Once these genes are cloned, the molecular mechanisms of resistance can be studied and durable strategies for resistance developed by genetic modification.

128 Conclusions

Acknowledgement

The gall midge is an economically important insect pest of rice occurring in many parts of the world including India, China, South-east Asian and African countries. It recurs annually in certain hotspots in India, causing a damage of 20–85% of the yield in these regions. At least five different biotypes of this pest have been identified in India and these biotypes are under a continuous state of evolution leading to the generation of new biotypes that cause the breakdown of resistance in rice. This makes it important to develop methods for identification of biotypes as well as to breed new rice varieties that provide resistance to the pre-existing as well as newly-emerging biotypes. Chemicals have not been very effective in controlling gall midge because of the location of larvae which lies within and is well protected by the gall. Besides, chemicals also pose environmental problems. Thus, deployment of resistance genes from resistant donors will not only be environment-friendly but also effective in providing durable resistance. However, monogenic resistance is not very long lasting. Therefore, deployment of many resistance genes would provide durable resistance against gall midge. Plant breeders have been consistently introgressing resistance genes from resistant donors, but this is a very long process involving many rounds of crossing and back crossing. Besides, screening lines for gall midge resistance in the field is problematic if favorable environmental conditions for the growth of gall midge do not exist. Therefore, development of a very effective marker assisted selection (MAS) protocol for the selection of resistant phenotypes would prove to be a boon for the plant breeders. Development of molecular markers tightly linked to gall midge resistance would help in pyramiding of resistance genes from many donors in a realistic time scale. At present, we are moving in this direction by working very closely with plant breeders and hopefully will be in a position to develop a system by which resistant rice varieties can be bred in a shorter time span. Also, different approaches are being taken, at present, to clone the gall midge resistance genes. Once these genes are cloned, they can be used for transformation of the susceptible elite cultivars with the many gall midge resistance genes available. The molecular mechanisms of resistance can also be studied and durable strategies for resistance evolved. This would help us to be a step ahead in the battle against an economically important insect pest of rice.

This work was supported in part by a grant from the Rockefeller Foundation.

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