Most pathosystems encompass intermeshed biological relationships between a pathogen and its hosts in a shared environment. If additional biological entities such as vectors of the pathogen are integral components, as with many plant virus systems, the complexity increases substantially. When the virus is widespread in several crop and perennial plant species, has a number of distinct variants, or the variants are spread selectively by several vector species, the complexity of the ecological interactions is even greater. All of these elements, when interacting concurrently, create exceptionally complex ecological pathosystems. The disease complex known as barley yellow dwarf epitomizes such systems. Investigating virus variants, their plant hosts and vectors, and elucidating the confounding resultant interactions in diverse and fluctuating environments in different regions of the world is an extremely difficult task, but one that must be undertaken to gain an understanding of the pathosystem. To do so

Epidemiology of Barley Yellow Dwarf: A Study in Ecological Complexity

Wheatgrasses (species of Agropyron complex) have previously been reported to be resistant to barley yellow dwarf virus (BYDV). To introgress this resistance into wheat, Triticum aestivum × Thinopyr...

Introgression and characterization of barley yellow dwarf virus resistance from Thinopyrum intermedium into wheat

SUMMARY

Seedlings of a series of addition or substitution lines of wheat containing different Thinopyrum intermedium chromosomes were inoculated with the PAV and RPV serotypes of barley yellow dwarf virus (BYDV). Reduced virus titres in infected plants were ascribed to a single pair of homoeologous group 7 chromosomes from Th. intermedium in the disomic addition lines L1 and TAF 2. The group 7 chromosome is associated with red pigmentation of coleoptiles, which was also observed in two lines ditelosomic for the α arm of the chromosome. However, when infected with the PAV serotype of BYDV, the ditelosomic lines had normal virus titres and it is concluded that potential determinants of BYDV resistance are located on the β arm of the Group 7 chromosome.

A single wheatgrass chromosome reduces the concentration of barley yellow dwarf virus in wheat

Genomic in situhybridization (GISH) to root-tip cells at mitotic metaphase, using genomic DNA probes from Thinopyrum intermedium and Pseudoroegneria strigosa, was used to examine the genomic constitution of Th. intermedium, the 56-chromosome partial amphiploid to wheat called Zhong 5 and disease-resistant derivatives of Zhong 5, in a wheat background. Evidence from GISH indicated that Th. intermedium contained seven pairs of St, seven JS and 21 J chromosomes; three pairs of Th. intermedium chromosomes with satellites in their short arms belonging to the St, J, J genomes and homoeologous groups 1, 1, and 5 respectively. GISH results using different materials and different probes showed that seven pairs of added Th. intermedium chromosomes in Zhong 5 included three pairs of St chromosomes, two pairs of JS chromosomes and two pairs of St-JS reciprocal tanslocation chromosomes. A pair of chromosomes, which substituted a pair of wheat chromosomes in Yi 4212 and in HG 295 and was added to 21 pairs of wheat chromosomes in the disomic additions Z1, Z2 and Z6, conferred BYDV-resistance and was identical to a pair of St-JS tanslocation chromosomes (StJS) in Zhong 5. The StJS chromosome had a special GISH signal pattern and could be easily distinguished from other added chromosomes in Zhong 5; it has not yet been possible to locate the BYDV-resistant gene(s) of this translocated chromosome either in the St chromosome portion belonging to homoeologous group 2 or in the JS chromosome portion whose homoeologous group relationship is still uncertain. Among 22 chromosome pairs in disomic addition line Z3, the added chromosome pair had satellites and belonged to the St genome and homoeologous group 1. Disomic addition line Z4 carried a pair of added chromosomes which was composed of a group-7 JS chromosome translocated with a wheat chromosome; this chromosome was different to 7 Ai-1, but was identical to 7 Ai-2. The leaf rust and stem rust resistance genes were located in the distal region of the long arm, whereas the stripe rust resistance gene(s) was located in the short arm or in the proximal region of the long arm of 7 Ai-2. A pair of JS-wheat translocation chromosomes, which originated from the WJS chromosomes in Z4, was added to the disomic addition line Z5; the added chromosomes of Z5 carried leaf and stem rust resistance but not stripe rust resistance; Z5 is a potentially useful source for rust resistance genes in wheat breeding and for cloning these novel rust-resistant genes. GISH analysis using the St genome as a probe has proved advantageous in identifying alien Th. intermedium in wheat.

Genomic in situ hybridization (GISH) analyses of Thinopyrum intermedium, its partial amphiploid Zhong 5, and disease-resistant derivatives in wheat.

Lophopyrum elongatum, a close relative of wheat, provides a source of novel genes for wheat improvement. Molecular markers were developed to monitor the introgression of L. elongatum chromosome segments into hexaploid wheat. Existing simple sequence repeats (SSRs) derived from genomic libraries were initially screened for detecting L. elongatum loci in wheat, but only 6 of the 163 markers tested were successful. To increase detection of L. elongatum specific loci, 165 SSRs were identified from wheat expressed sequence tags (ESTs), where their chromosomal positions in wheat were known from deletion bin mapping. Detailed sequence analysis identified 41 SSRs within this group as potentially superior in their ability to detect L. elongatum loci. BLASTN alignments were used to position primers within regions of the ESTs that have sequence conservation with at least 1 similar EST from another cereal species. The targeting of primers in this manner enabled 14 L. elongatum markers from 41 wheat ESTs to be identified, whereas only 2 from 124 primers designed in random positions flanking SSRs detected L. elongatum loci. Addition and ditelosomic lines were used to assign all 22 markers to specific chromosome locations in L. elongatum. Nine of these SSR markers were assigned to homoeologous chromosome locations based on their similar position in hexaploid wheat. The remaining markers mapped to other L. elongatum chromosomes indicating a degree of chromosome rearrangements, paralogous sequences and (or) sequence variation between the 2 species. The EST-SSR markers were also used to screen other wheatgrass species indicating further chromosome rearrangements and (or) sequence variation between wheatgrass genomes. This study details methodologies for the generation of SSRs for detecting L. elongatum loci.

EST-derived SSR markers from defined regions of the wheat genome to identify Lophopyrum elongatum specific loci.

Shukle, R. H .• Lampe, D. J ., Lister, R. M .• and Foster, J. E. 1987. Aphid feeding behavior: Relationship to barley yellow dwarf vi rus resistance in Agropyron species. Phytopathology 77: 725-729. The infectibility of various Agropyron species (wheatgrasses) was examined with respect to thl\"« isotates of barley yellow dwarf virus (BYDV) by infl:$ting them with appropriate vector aphids, followed by testing by enzyme-linked immunosorbent assay. Feeding behavior of the VCi:tors Rhopalosiphum padi and SilObion avenoe was also electronically monitored to determine their abi lity to inoculate phloem. The results indicated that resistance to BYDV infection occurs in several Agrop>'n}n species. For most species tested. resistance seemed due to failure in virus increase. but in $Orne species a major constraint on infection \\\\'as the inability ofvcctors 10 locate phloem. Two potential approaches to breeding for reduced BYDV in wheat by crossing with Agropyron species may thus be: incorporating facto rs reducing or preventing virus production and incorporating factors reducing the ability of \"ectors to inoculate plants successfully. Addirlonal kry words: 8 YDV-resistant germ plasm.luteoviru.ses, wheat improvement. The term barley yellow dwarf virus (BYDV) includes at least five variously interrelated types of luteovirusts (9. 18) that are appa rently res t ricted to gramineou5 plants. They cause yellowdwarf diseases in cereals that affect the su pply of whcat, oats, and barley throughout the world (6, 18). Like the other luteovirusts, Th. publlc'!ion COlt. of !ht •• nlcle wer. def,.yed In p.,t by p.~ chl'g. p.yment This .,Uct. mu\" lI,or.r.,..o be horoby mlrked \" • .,.. ... ,; ..... .\",In _,,.nco wllh t8 U.S.C. t 11304 s<MIy 10 Indlco!. tN. 'Ke. Thl, artlcte I. In the public domain and not copyrightable. It may be ,reely reprin ted with (u,tomery crediting o r the .ource. The American PhytOp .. hotoglcat Society, t981. they arc obligatorily transmi tted by aphids in a circulative manner and are rest ricted to phloem. Two important vectors of BYDV are the English grain aphid. SilObion Qvenae F. , and the bird cherry oat aphid , Rhopafosiphum pad; L. S. avenal' transmits the MAV and PAY isolates (sensu Rochow, 17), whereas R.paditransmitsthe PAVand RPV isolates ( 17). Recent resul ts in experiments with electronic monitoring of feeding behavior have provided direct evidence that penetration of phloem sieve elements is a prerequisi te for BYDV tra nsmission by these vectors (19. Lampe et al,lInpublished). Infection with BYDV might therefore be avoided in cereal cul tivan by resistance to phloem contact and ingestion. Vol. 77. No.5, 1987 725 Currently. tolerancc (sensu Cooper and Jones. 3) for BYDV infection occurs in wheat. Triticum at'slivum L. em. Theil. ( 14.15), but there arc no clear-cut examples of immunity or of the resistance (rcduccd virus production) that occur in some cultivars of ba rley (16). If such were available in wheat, or in species that can intercross with wheat, it could have considerable significance for BYDV resistance brecding programs. In this regard, high levels of resistance (i.e., virus could not be detected by cnzyme-linked immunosorbcnt assay [ELISA] in plants infestcd with vi ruliferous aphids) were recently rcported with respect to two BYDV isolates, PAY and RMV. in several Agropyron species (23). Thcre is, however, no information concerning such resistance with other isolates of BYDV. or whcthcr it may actually reflect the inability of vectors to inoculate plants efficiently. The objectives of the present study were: to determine if va rious Agropyron species show high levels of resistance to virus product ion with a PAY-like isolate of BYDV ( P-PAV, 7) or with the MAV and RPV isolates of BYDVand to electronically monitor the feeding behavior of aphids on the Agropyron species to determine whether they are readily able to locate and ingcst from phloem tissue. MA TERIALS AND METH ODS Insects and virus isolates. Viruliferous R. padi carrying the P-PAVor RPV isolates and S. aVt'nut' carrying the MAY isolate of BYDY. respectively. were obtained from cultures maintained on infected oats. A vena saliva L. 'Clintland 64', in isolated chambers at 21 C with a l6-hr photoperiod. Viruliferous aphids used to infest Agropyron species and oats and to monitor feeding behavior were apterous, vi rginoparous adults. The RPY and MAV isolates were subcultures maintaincd from cultures initially supplied by Dr. W. F. Rochow (24): the PP A V isolate has been described (7). P lants. Seedling plants of s ix wheatgrasses, Agropyro n imerme,/ium (Host) Beauv., Agropyron elongalllll1 (Host) Beauv. 'Jose', Agropyron sibiricum (WilJd.) Beauv .. and Agrop)'ron smithii Rydb . 'Arriba', 'B.1Tton', and 'Rosanna' were grown from seed under greenhouse conditions beneath sodium lights (930 ~ Einsteinsl mll sec) with a 16-hr photoperiod. Seeds of the Agropyro\" species were obtained from commercial sources. Plants were grown in sterile soil mix and irrigated with water containing a soluble N PK ferti lizer ( 15-30-15). Tests for BY DV infectibility and aphid fceding behavior were conducted with seedli ng plants in the third-leaf stage (1418 days after emergence). Tests for infeclibility with 8 YDY Isolates. Plants were tested for infeetibili ty with the BYDV isolates by infestation with 10 vi ruliferous, adult aphids (vide supra) per plant. Ten plants were used for each Agropyron species tested. Infested plants were maintained in isolation at 21 C (16-h r photoperiod) for I wk. Aphid survival was recorded and aphids were then killed by spraying wit h malathion. The plants were then grown for an additional 14 days under greenhouse conditions. Noni nfested control plants and infested Clintland 64 oats were treated si milarly. Tissue samples for EL ISA consisted of each entire plant (including roots). washed clean and dried by blotting. Extraction lind ELISA. Plant samples were first pulverized ill liquid nitrogen in a mortar. Potassium phosphate buffer (0.1 M, pH 7) containing 0.05% Tween-20 and 2% polyvinyl pyrrolidone (MW 40,000) was then added (1:6, w/ v) along with a pinch of Carborundum, and the tissue ground further. The resulting extract was filtered through cheesecloth and stored for 12 wk at 76 C until assayed for virus. Virus in extracts was assayed by ELISA as described by Hammond et al (7) with the modifications of Skaria et al (24). Monitorinc . phid feeding behavior. Aphid fceding behavior was electronically monitored using a system similar to that described by McLean and Kinsey (10,11). In our system (19). a 12-~m gold wire attached to the dorsum of the aphid with silver conducting paint serves as one electrode. After a I-hr starvation period, the aphid is given access to the leaf ofa plant and a 20 mY, 330 H\"I. signal is transmitted via the electrode attached to the aphid to a 726 PHYTOPATHOLOGY ground implanted in the potting med ium. During salivation and ingestion th e aphid se rves as a variable impedance and modulations of the signal arc amplified and recorded. Th is technique has been used to monitor the feeding behavior of several aphid species (2,13,19). Three basic waveform patterns are recorded using the system: an \"S~-waveform corresponding to salivation and sheath-formation, an \"X \"-waveform corresponding to style t penetration of sieve elements, and an \" I\" waveform corresponding to a conti nuo us period of ingestion . Any combination of sequences of the three basic waveforms can occur during aphid feeding: however, an I-wave immediately preceded by an X-wave, or a series of X-waves, is considered to be diagnostic of phloem ingestion ( 11 , 19). To record feeding behavior on Agropyron species. an individual aphid was placed on the second or third leafofa seedling pla nt and feedi ng activities recorded for 180 min from the start of the first probe. All recordings were made in the laboratory at room temperature (22 q. For each Agropyron species, feeding behavior wa s recorded for six S. ave\"ae and six R. padi on individual seedlings. The following events associated with aphid feeding were recorded: number of probes, salivation, number of phloem contacts, phloem ingestion, no n phloem ingestio n, and nonprobing. Histologlnl .nod.tion of stylet t ip location. Association of style t tip location with reco rd ed wavefo r ms was done histologically for S. avt'llae and R. padi feeding on Clint land 64 oats and A. in/ermedium . The procedure was similar to that described by Mc Lean and Kinsey (I I). At the occurrence of a waveform to be investigated, the aphid wa s anesthct ized wi th COl. The wire connecting the aphid to the feeding monitor was then severed , and a leaf segment with the aphid in position was cut from the blade and placed into a Craf fixative containing 5% nicoti ne and 0. 1% Triton x-loa. This solution rap idly killed the anesthetized aphid. After 2 min, the leaf segment with the aphid in position was removed, trimmed to the desired size, and placed into a Cmf fixa tive lacking the nicotine and Triton XIOO. After fixation for 12-24 hr at 4 C, the tissue was dehydra ted by passage through an et hanol series. The tissue was then embedded in Paraplast and 10 -~m sections cut. Throughout dehydration and embedding the aphid was left in position on the leaf segment. Sections were then stained with safranin and fa st green. and the posit ion of the aphid's stylets in the plant tissue was associated with the corresponding waveform.

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David J. Lampe

Papers

Aphid Feeding Behavior: Relationship to Barley Yellow Dwarf Virus Resistance in Agropyron Species