A novel wheat gene encoding a putative chitin-binding lectin is associated with resistance against Hessian fly

The gene-for-gene interaction triggering resistance of wheat against first-instar Hessian fly larvae utilizes specialized defence response genes not previously identified in other interactions with pests or pathogens. We characterized the expression of Hfr-3 , a novel gene encoding a lectin-like protein with 68–70% identity to the wheat germ agglutinins. Within each of the four predicted chitin-binding hevein domains, the HFR-3 translated protein sequence contained five conserved saccharide-binding amino acids. Quantification of Hfr-3 mRNA levels confirmed a rapid response and gradual increase, up to 3000-fold above the uninfested control in the incompatible interaction 3 days after egg hatch. Hfr-3 mRNA abundance was influenced by the number of larvae per plant, suggesting that resistance is localized rather than systemic. In addition, Hfr-3 was responsive to another sucking insect, the bird cherry-oat aphid, but not to fall armyworm attack, wounding or exogenous application of methyl jasmonate, salicylic acid or abscisic acid. Western blot analysis demonstrated that HFR-3 protein increased in parallel to mRNA levels in crown tissues during incompatible interactions. HFR-3 protein was detected in both virulent and avirulent larvae, indicating ingestion. Anti-nutritional proteins, such as lectins, may be responsible for the apparent starvation of avirulent first-instar Hessian fly larvae during the initial few days of incompatible interactions with resistant wheat plants.     

Genotypic interaction between resistance genes in wheat and virulence genes in the Hessian fly Mayetiola destructor (Diptera: Cecidomyiidae)

The genotypic interaction between wheat resistance genes H3, H6, H7H8, H9 and virulence genes vH3, vH6, vH7vH8, vH9 of Hessian fly, Mayetiola destructor (Say), was studied in a growth chamber. Results showed that plants homozygous and heterozygous for the H3 gene expressed a high level of resistance against homozygous avirulent and heterozygous larvae carrying the vH3 virulence allele. The H7H8 genes were highly effective in the homozygous condition, but displayed a reduced level of resistance in the heterozygous condition. The H6 and H9 genes showed different levels of resistance against the reciprocal heterozygous larvae (vH6(a)vH6(A) versus vH6(A)vH6(a) and vH9(a)vH9(A) versus vH9(A)vH9(a)). Adults reared from vH6(a)vH6(A) and vH9(a)vH9(A) larvae were all males, consistent with the vH6 and vH9 X-linkage. Plants homozygous for H3, H6, H7H8, and H9 allowed for greater larval survival of heterozygous larvae, which suggests that avirulence to these resistance genes is incompletely dominant. Greater survival of homozygous avirulent larvae on heterozygous plants (H3h3, H6h6, H7h7H8h8, H9h9) suggests incomplete dominance of these wheat genes. Survival of heterozygous along with homozygous virulent larvae would reduce selection pressure for virulence in Hessian fly populations infesting fields of resistant wheat cultivars. This would be expected to slow the increase in frequency of virulence alleles that often results from deployment of resistant cultivars.     

Avoidance of occupied hosts by the Hessian fly: oviposition behaviour and consequences for larval survival

Abstract 1. Unoccupied wheat plants and wheat plants occupied by conspecific eggs or larvae were presented to ovipositing female Hessian flies in choice tests.
2. The presence of conspecific eggs on the leaf surfaces of wheat plants did not appear to have any effect on the responses of ovipositing Hessian fly females.
3. The presence of conspecific larvae at the base and nodes of wheat plants for 1, 6, or 11 days had significant effects on Hessian fly oviposition. Eggs oviposited on plants were inversely proportional to larval densities and days of larval occupation.
4. Feeding by Hessian fly larvae is associated with several changes in wheat plants. One of these changes, the growth arrestment of the plant, was measured by recording the heights of plants used in oviposition tests. Plant heights were inversely proportional to both larval densities and days of occupation. Plant heights were directly proportional to eggs oviposited on plants.
5. The consequences of adult female avoidance of plants occupied by conspecific larvae were investigated by allowing females to oviposit on unoccupied plants and 1-day, 6-day, and 11-day larval occupied plants, then scoring at the end of the first larval instar the survival of the offspring that resulted from this oviposition.
6. Survival during the first larval instar was 88% for the offspring of females that oviposited on unoccupied plants, decreasing to 82, 31, and 4% on the 1-day, 6-day, and 11-day occupied plant treatments. On these four plant treatments, a positive correlation was found between larval performance (i.e. survival) and the preferences of ovipositing females.
7. On the four plant treatments, relationships between first-instar larval density and first-instar larval survival varied significantly. On unoccupied plants, survival was inversely proportional to density. On plants oviposited on at 6 days of larval occupation, survival was directly proportional to density.     

Gene-for-gene defense of wheat against the Hessian fly lacks a classical oxidative burst

Genetic similarities between plant interactions with microbial pathogens and wheat interactions with Hessian fly larvae prompted us to investigate defense and counterdefense mechanisms. Plant oxidative burst, a rapid increase in the levels of active oxygen species (AOS) within the initial 24 h of an interaction with pathogens, commonly is associated with defenses that are triggered by gene-for-gene recognition events similar to those involving wheat and Hessian fly larvae. RNAs encoded by Hessian fly superoxide dismutase (SOD) and catalase (CAT) genes, involved in detoxification of AOS, increased in first-instar larvae during both compatible and incompatible interactions. However, mRNA levels of a wheat NADPH oxidase (NOX) gene that generates superoxide (O2-) did not increase. In addition, inhibiting wheat NOX enzyme with diphenyleneiodonium did not result in increased survival of avirulent larvae. However, nitro blue tetrazolium staining indicated that basal levels of O2- are present in both uninfested and infested wheat tissue. mRNA encoded by wheat genes involved in detoxification of the cellular environment, SOD, CAT, and glutathione-S-transferase did not increase in abundance. Histochemical staining with 3,3-diaminobenzidine revealed no increases in wheat hydrogen peroxide (H2O2) during infestation that were correlated with the changes in larval SOD and CAT mRNA. However, treatment with 2',7'-dichlorofluorescin demonstrated the presence of basal levels of H2O2 in the elongation zone of both infested and uninfested plants. The accumulation of a wheat flavanone 3-hydroxylase mRNA did show some parallels with larval gene mRNA profiles. These results suggested that larvae encounter stresses imposed by mechanisms other than an oxidative burst in wheat seedlings.     

Biotype composition of Hessian fly (Diptera: Cecidomyiidae) populations from the southeastern, midwestern, and northwestern United States and virulence to resistance genes in wheat

Twenty-three Hessian fly, Mayetiola destructor (Say), populations collected in the southeastern (Alabama and Mississippi), midwestern (Indiana), and northwestern (Idaho and Washington) United States from 1995 to 1999 were evaluated for biotype composition based on response to Hessian fly resistance genes H3, H5, H6, and H7H8 in wheat, Triticum aestivum L. Biotypes L and O, combined, made up at least 60% of all Alabama populations. Biotype L was predominant in the northern third of Alabama and biotype O in the southern two-thirds of the state. Based on biotype data, wheat cultivars with H7H8 resistance should be highly effective in central and southern Alabama. Fifty-four percent of the Mississippi population consisted of biotype L, and the remaining virulent biotypes (B, D, E, G, J, and O) ranged in frequency from 1 to 17%. The Mississippi population also contained 4% of the avirulent biotype GP. Only biotypes D and L were found in Indiana populations, but biotype L was predominant. Hessian fly populations from Idaho and Washington contained one or more of the virulent biotypes D-H, J, and L-O; however, only biotypes E, F, and G occurred at frequencies > 12%. The avirulent biotype GP made up 25-57% of Idaho and Washington populations, a much higher percentage than found in populations from the eastern United States. Although the highest level of virulence in Idaho and Washington populations was found to resistance genes H3 and H6, the frequency of biotype GP would indicate that the currently deployed gene H3 would provide a moderate to high level of resistance, depending on location. Nine of the populations, plus populations collected from the mid-Atlantic state area in 1989 and 1996, also were tested against the wheat cultivar 'INW9811' that carries H13 resistance to Hessian fly biotype L and two Purdue wheat lines with unidentified genes for resistance. The H13 resistance in INW9811 was highly effective against all populations tested from the eastern and northwestern U.S. wheat production areas, except Maryland and Virginia. Population studies also indicated that wheat line CI 17960-1-1-2-4-2-10 likely carries the H13 resistance gene, based on the similarity of its response and that of INW9811 to eight fly populations. Continued monitoring of biotype frequency in Hessian fly populations is required for optimal deployment and management of resistance genes in all wheat production areas.     

Feeding by Hessian fly [Mayetiola destructor (Say)] larvae does not induce plant indirect defences

Abstract.  1. Recent research has addressed the function of herbivore-induced plant volatiles in attracting natural enemies of feeding herbivores. While many types of insect herbivory appear to elicit volatile responses, those triggered by gall insects have received little attention. Previous work indicates that at least one gall insect species induces changes in host-plant volatiles, but no other studies appear to have addressed whether gall insects trigger plant indirect defences.
2. The volatile responses of wheat to feeding by larvae of the Hessian fly Mayetiola destructor (Say) (Diptera: Cecidomyiidae) were studied to further explore indirect responses of plants to feeding by gall insects. This specialist gall midge species did not elicit a detectable volatile response from wheat plants, whereas a generalist caterpillar triggered volatile release. Moreover, Hessian fly feeding altered volatile responses to subsequent caterpillar herbivory.
3. These results suggest that Hessian fly larvae exert a degree of control over the defensive responses of their host plants and offer insight into plant-gall insect interactions. Also, the failure of Hessian fly larvae to elicit an indirect defensive response from their host plants may help explain why natural enemies, which often rely on induced volatile cues, fail to inflict significant mortality on M. destructor populations in the field.     

Treatment of adult Coleoptera with a chitin synthesis inhibitor affects mortality and development time of their progeny

A group of insect growth regulators, the chitin synthesis inhibitors (CSIs), are being more widely used as many insects have become resistant to broad-spectrum insecticides. This study investigated the mode of action of one CSI, particularly looking at the delayed effect of treating adult insects on the survival and development of their progeny. The study describes the responses of adult stored grain beetles Sitophilus oryzae (L.) (Curculionidae) and Rhyzopertha dominica (F.) (Bostrichidae) to wheat treated with chlorfluazuron. For both species, wheat treated with chlorfluazuron reduced progeny survival at a constant rate from 1 day to 8 weeks after it had been treated. There was a direct, negative concentration-time relationship between the treatment of the adults with chlorfluazuron-treated wheat and the survival and developmental rate of their progeny. Adults that were pre-exposed for one or more weeks before oviposition to wheat treated with chlorfluazuron had significantly lower progeny survival than adults that were not pre-exposed. The EC₉₅ values were 16-fold higher for R. dominica and 40-fold higher for S. oryzae from assays without pre-exposure, than with pre-exposure. Pre-exposure of R. dominica with 0.1 mg kg^-1 and of S. oryzae with 0.4 mg kg^-1 of chlorfluazuron reduced progeny survival by 95%. Higher concentrations of chlorfluazuron produced a reduction in mean population developmental time of several days. These findings have important implications for bioassays of CSIs, since pre-exposure of the adults can significantly reduce the numbers of F1 progeny. Therefore standard bioassays may seriously underestimate the efficacy of the CSI being assayed.     

Feeding by Hessian Fly (Mayetiola destructor [Say]) Larvae on Wheat Increases Levels of Fatty Acids and Indole-3-Acetic Acid but not Hormones Involved in Plant-Defense Signaling

Gall-inducing insects exert a unique level of control over the physiology of their host plants. This control can extend to host–plant defenses so that some, if not most, gall-inducing species appear to avoid or modify host plant defenses to effect production of their gall. Included among gall insects is Hessian fly (Mayetiola destructor [Say], Diptera: Cecidomyiidae), a damaging pest of wheat (Triticum aestivum L.) and an emerging model system for studying plant–insect interactions. We studied the dynamics of some defense-related phytohormones and associated fatty acids during feeding of first instar Hessian fly larvae on a susceptible variety of wheat. We found that Hessian fly larvae significantly elevated in their host plants’ levels of linolenic and linoleic acids, fatty acids that may be nutritionally beneficial. Hessian fly larvae also elevated levels of indole-3-acetic acid (IAA), a phytohormone hypothesized to be involved in gall formation, but not the defense-related hormones jasmonic (JA) and salicylic acids. Moreover, we detected in Hessian fly-infested plants a significant negative relationship between IAA and JA that was not present in control plants. Our results suggest that Hessian fly larvae may induce nutritionally beneficial changes while concomitantly altering phytohormone levels, possibly to facilitate plant-defense avoidance.     

Interaction of treatment of both adult and immature Coleoptera with a chitin synthesis inhibitor affects mortality and development time of their progeny

Chitin synthesis inhibitors, like many other insect growth regulating insecticides, do not kill adult insects but cause mortality of the immature stages. Pre-exposure of adult stored grain Coleoptera, Rhyzopertha dominica (F.) (Bostrichidae) and Sitophilus oryzae (L.) (Curculionidae) and development of their progeny in grain treated with the chitin synthesis inhibitor, chlorfluazuron, influenced the mortality and development rate of the progeny. Hatch rate of eggs from R. dominica adults that had both developed and laid on wheat treated with 0.75 mg kg–1 chlorfluazuron was reduced by almost 50% compared with untreated eggs, with an LC50 of 0.84 mg kg–1 . Eggs laid on treated wheat by R. dominica adults that had been exposed only to treated wheat for 2 weeks before oviposition showed greater reduction in hatch: 75% reduction of normal hatch rate at 0.25 mg kg–1 and almost 100% reduction at 2 mg kg–1 chlorfluazuron, with an LC50 of 0.19 mg kg–1 chlorfluazuron. X-rays were used to monitor the development and mortality of the immature stages of S. oryzae that developed within the wheat grains. Numbers of eggs laid were not affected by chlorfluazuron treatment. The combination of pre-exposure of adults and chlorfluazuron concentration had an additive effect on mortality of immature S. oryzae. Pre-exposure of adults caused most mortality in the first three weeks of development (eggs and larvae), whereas development in treated wheat caused mortality from weeks 3 to 8 (pupae and adults); higher concentrations of chlorfluazuron caused higher mortality. Development in wheat treated with 1 mg kg–1 chlorfluazuron caused 12% corrected overall mortality of progeny while pre-exposure to the same concentration and development in untreated wheat caused 29% corrected mortality. Pre-exposure combined with development in wheat treated with 1 mg kg–1 caused 30% corrected mortality. Thus, pre-exposure of adults appears to have a greater effect on mortality of S. oryzae progeny than development of immature stages in treated grain. Development on treated grain had no effect on development rate. Pre-exposure of adults did not appear to affect the rate of immature development, as assessed by X-rays, but did slow the emergence of adults, lengthening development time by about 2 days. This significant, additive effect of pre-exposure of adults on the mortality of their progeny will enhance the toxicity of chitin synthesis inhibitors such as chlorfluazuron, since most adults receive treatment when the immature stages are treated in crops either before they are harvested or in storage. Assessing the proportion of eggs that hatch from pre-exposed adults would be a simpler bioassay for CSIs.     

Does R gene resistance allow wheat to prevent plant growth effects associated with Hessian fly (Diptera: Cecidomyiidae) attack?

Resistance genes (R genes) are an important part of the plant's immune system. Among insects, the Hessian fly, Mayetiola destructor (Say) (Diptera: Cecidomyiidae), larva is the target of the greatest number of characterized R genes (H1-H32). The biochemical/molecular mechanism of R gene resistance to Hessian fly is not well understood. In the absence of an effective R gene, larvae caused extensive growth deficits (> 30 cm) in wheat seedlings. In the presence of one of three effective R genes, H6, H9, or H13, larvae caused small growth deficits (approximately 3-4 cm) in two leaves (third and fourth) that were actively growing during the first days of larval attack. After larvae died on R gene plants, the fifth leaf and tiller leaves exhibited small increases in growth (2-4 cm). Growth responses of susceptible and resistant plants diverged at a time when Hessian fly larvae were establishing a nutritive gall tissue at feeding sites. The results of this study support the hypothesis that R gene resistance cannot prevent initial larval attack, but, by stopping the formation of the larval gall, it prevents the most serious consequences of larval attack.     

Ultrastructural changes in the midguts of Hessian fly larvae feeding on resistant wheat

The focus of the present study was to compare ultrastructure in the midguts of larvae of the Hessian fly, Mayetiola destructor (Say), under different feeding regimens. Larvae were either fed on Hessian fly-resistant or -susceptible wheat, and each group was compared to starved larvae. Within 3 h of larval Hessian fly feeding on resistant wheat, midgut microvilli were disrupted, and after 6 h, microvilli were absent. The disruption in microvilli in larvae feeding on resistant wheat were similar to those reported for midgut microvilli of European corn borer, Ostrinia nubilasis (Hubner), larvae fed a diet containing wheat germ agglutinin. Results from the present ultrastructural study, coupled with previous studies documenting expression of genes encoding lectin and lectin-like proteins is rapidly up-regulated in resistant wheat to larval Hessian fly, are indications that the midgut is a target of plant resistance compounds. In addition, the midgut of the larval Hessian fly is apparently unique among other dipterans in that no peritrophic membrane was observed. Ultrastructural changes in the midgut are discussed from the prospective of their potential affects on the gut physiology of Hessian fly larvae and the mechanism of antibiosis in the resistance of wheat to Hessian fly attack.     

Host plant quantitative trait loci affect specific behavioral sequences in oviposition by a stem-mining insect

Key message

Genetic diversity in quantitative loci associated with plant traits used by insects as cues for host selection can influence oviposition behavior and maternal choice.

Abstract

Host plant selection for oviposition is an important determinant of progeny performance and survival for phytophagous insects. Specific cues from the plant influence insect oviposition behavior; but, to date, no set of host plant quantitative trait loci (QTLs) have been shown to have an effect on behavioral sequences leading to oviposition. Three QTLs in wheat (Triticum aestivum L.) have been identified as influencing resistance to the wheat stem sawfly (WSS) (Cephus cinctus Norton). Wheat near-isogenic lines (NILs) for each of the three QTLs were used to test whether foraging WSS were able to discriminate variation in plant cues resulting from allelic changes. A QTL on chromosome 3B (Qss-msub-3BL) previously associated with stem solidness and larval antibiosis was shown to affect WSS oviposition behavior, host preference, and field infestation. Decreased preference for oviposition was also related to a QTL allele on chromosome 2D (Qwss.msub-2D). A QTL on chromosome 4A (Qwss.msub-4A.1) affected host plant attractiveness to foraging females, but did not change oviposition preference after females landed on the stem. These findings show that oviposition decisions regarding potential plant hosts require WSS females to discriminate signals from the plant associated with allelic variation at host plant quantitative loci. Allele types in a host plant QTL associated with differential survival of immature progeny can affect maternal choices for oviposition. The multidisciplinary approach used here may lead to the identification of plant genes with important community consequences, and may complement the use of antibiosis due to solid stems to control the wheat stem sawfly in agroecosystems.

     

No fitness cost for wheat's H gene-mediated resistance to Hessian fly (Diptera: Cecidomyiidae)

Resistance (R) genes have a proven record for protecting plants against biotic stress. A problem is parasite adaptation via Avirulence (Avr) mutations, which allows the parasite to colonize the R gene plant. Scientists hope to make R genes more durable by stacking them in a single cultivar. However, stacking assumes that R gene-mediated resistance has no fitness cost for the plant. We tested this assumption for wheat's resistance to Hessian fly, Mayetiola destructor (Say) (Diptera: Cecidomyiidae). Our study included ten plant fitness measures and four wheat genotypes, one susceptible, and three expressing either the H6, H9, or H13 resistance gene. Because R gene-mediated resistance has two components, we measured two types of costs: the cost of the constitutively-expressed H gene, which functions in plant surveillance, and the cost of the downstream induced responses, which were triggered by Hessian fly larvae rather than a chemical elicitor. For the constitutively expressed Hgene, some measures indicated costs, but a greater number of measures indicated benefits of simply expressing the H gene. For the induced resistance, instead of costs, resistant plants showed benefits of being attacked. Resistant plants were more likely to survive attack than susceptible plants, and surviving resistant plants produced higher yield and quality. We discuss why resistance to the Hessian fly has little or no cost and propose that tolerance is important, with compensatory growth occurring after H gene-mediated resistance kills the larva. We end with a caution: Given that plants were given good growing conditions, fitness costs may be found under conditions of greater biotic or abiotic stress.     

Oviposition preferences of the Hessian fly and their consequences for the survival and reproductive potential of offspring

1. The relationship between oviposition preference and offspring performance was investigated experimentally for the Hessian fly Mayetiola destructor (Say) using 10 grass genotypes that represented five different genera and six different species. Oviposition preferences were quantified in a choice test using arrays of 200–400 plants. Offspring performance was estimated by recording survival during three phases of the insect–plant association and by measuring wing length, a correlate of adult reproductive potential. Density effects were examined for all offspring variables, and were taken into account when offspring performance was compared across the 10 grasses. 2. Egg counts from the oviposition choice test revealed a consistent ranking of plants: 18ITSN triticale > Otane bread wheat = Caldwell bread wheat = Stacy bread wheat > 3424 bread wheat = PND durum wheat = Fleet barley = Valetta barley = Matua brome grass > Awapuni oat. 3. Survival and adult wing length varied significantly on the 10 plant types. Survival was ranked: Otane bread wheat > 3424 bread wheat = Fleet barley = Matua brome grass > Caldwell bread wheat = Valetta barley > 18ITSN triticale = PND durum wheat = Stacy bread wheat > Awapuni oat. Adult male and female wing lengths were greatest on Otane, the bread wheat that also provided the best survival. 4. For Hessian flies on each of the 10 plant types, data on survival, wing length, and wing length–fecundity relationships were combined into a single fitness measure. When these fitness measures were compared with egg counts, no overall pattern emerged. On seven of the 10 plant types, there was a positive linear relationship between egg counts and offspring fitness. On the other three plant types, egg counts were high while fitness was low. Possible reasons why Hessian fly females lay eggs on plants that are poor hosts for their offspring are discussed.