II. Newsletter Contributions

BYDV tolerance and multiple disease resistance in Triticum durum interspecific derivatives

A. Comeau¹⁾, C. A. St-Pierre²⁾, B. Kol²⁾, S. Pouleur¹⁾, and R. Paquet¹⁾

1) Agriculture and Agri-Food Canada, Sainte-Foy, Quebec G1V 2J3, Canada; 2) Dept. Phytologie, Universite Laval, Sainte-Foy, Quebec G1K 7P4, Canada

Over the last 20 years, Triticum durum has always been quoted as more sensitive to BYDV than bread wheat, Triticum aestivum. From 1981 to 1991 our own efforts aimed at developing BYDV resistant germplasm, met with very little success in durum wheat. The durum line 82 PCD-476 was chosen as the most reliable moderately tolerant check. It was selected after 10 years of trials but it was much inferior in tolerance to Maringa bread wheat or any barley possessing the Yd2 gene.

In Quebec, the climate is often humid, and durum wheat develops a broad range of other disease problems in those conditions, including scab (Fusarium head blight), root rot and other fungal diseases.

Tolerance vs. resistance
Recognizing that tolerance differs from resistance, we attempted in 1983-86 to see if resistance genes, selectable by ELISA, would allow faster progress. Alien sources contain high resistance (seen as low ELISA values) and also good tolerance to BYDV. Alien introgression was attempted as a way to improve tolerance and resistance of bread and durum wheat faster. The field trials on yield losses would often reveal that non-resistant wheat had quite good tolerance.

Fig.1. Seed quality comparison                      Fig. 2. Vigor of BYDV
(visual) in a year of scab                                   inoculated plants
epidemic, 1996 (1996)

One of the best tolerant lines was an interspecific-derived Triticum aestivum line from China, Long Miai 10, which is putatively derived from wheat x Thinopyrum intermedium. Interestingly enough, Long Miai 10 had ELISA levels generally higher than Maringa bread wheat (let us note Maringa also has rather high ELISA values, and is used as BYDV tolerant check in our trials).

Introgression process
This line, Long Miai 10, was therefore used as a parent in crosses to both T aestivum and T. durum. The progenies were backcrossed to T aestivum and T. durum, selfed, and selected during the segregating generations F3-F5 using artificial BYDV inoculations. The first three cycles of selection of the T. durum trispecific populations showed very little promise. Out of about 8000 plants per year, not a single one seemed superior to 82 PCD-476 for BYDV tolerance. Then in the summer of 1993, about 10 plants that looked like "escapes" were harvested, from a cross labeled 88ADD-11. In this cross, line 82 PCD-476 is the recurrent durum parent. The good looking plants were multiplied for replicated trials.

In 1994 and 1995, the rows showed vigor and general disease resistance. In 1996, contrast was extreme between all the conventional durum wheat, which was severely affected by BYDV, and the 88ADD-11 re-selections which were as tolerant as Yd2 barleys (Figs. 1, 2, 3). The 88ADD-11 series also showed better scab resistance than conventional T. durum in 1996. In 1997, the germination was uneven due to drought, but still the "88ADD-11" interspecific lines were the best BYDV tolerant (Fig. 4).

Fig. 3. Yield and symptoms of BYDV inoculated lines of T. durum in 1996, compared to T. aestivum Anza (mod. tol.), and Atlas 68 barley (resistant). The "88ADD-11" lines had low symptoms and high yield.

Fig. 4. Yield and symptoms of BYDV inoculated lines of T. durum in 1997, compared to T. aestivum Maringa (tolerant). The "88ADD-11" lines had low symptoms and high yield.

Observations on roots
The root system of the interspecific durum lines seemed more healthy than that of conventional durum, and visual inspection of roots revealed fewer root rot lesions. We are increasingly aware that one effect of artificial BYDV selection is to improve root health in many ways. But in this case, the root health effect seemed more important and desirable, as all conventional durum wheat lines become very sensitive to root rot by flowering time, in our growing area.

We also wondered if the root morphology might be different, and evaluated this hypothesis in tests done in long acrylic tubes, 1.3 meter long x 8 cm wide, with virus-free and BYDV-inoculated plants. Roots were washed at harvest and the root distribution was observed. Susceptible check Kyle had overall a slightly larger biomass of roots throughout the length of the tubes. All lines had a reduction of roots from BYDV infection. However, the interspecific line kept a better capacity of branching in the 50-69 cm deep area, which led to a maintenance of root mass despite the BYDV infection (Fig. 5). Some root rot, probably seed-transmitted, was visible on roots of 82PcDuros-476. We feel that more studies on root health are needed to better understand the observed performance of durum germplasm in BYDV inoculated conditions.

Fig. 5. Ratio of "BYDV infected / check" root biomass at various depth for susceptible durum Kyle and two BYDV tolerant durum lines.

Conclusion
Our current observations on BYDV, root rot and scab confirm that the cross 88 ADD-11 has yielded, after a long, tedious selection process involving artificial infection of segregating populations with BYDV, a very small percentage of exceptional disease-tolerant germplasm. This germplasm may be useful in breeding durum wheat with better disease resistance, which might help to change the area of adaptation of this species, and for example make it more adapted in Manitoba, where BYDV and scab are more common. The segregation of resistance and potential usefulness of the 88 ADD-11 lines should be evaluated in test crosses to cultivated durum lines.

Study and identification of markers of two genes for resistance to BYDV derived from Thinopyrum intermedium

C. Etienne, J. Jahier, D. Barloy and M. Trottet

INRA, Station d’Amélioration des Plantes, BP 29, 35653 Le Rheu, France

Two genes of partial resistance derived from Thinopyrum intermedium have been studied. The first one derived from an addition line of chromosome 7Agⁱ in a Vilmorin 27 background extracted from a backcross progeny of a partial amphiploid (TAF46, 2n=56), created by Y. Cauderon. A translocation 7D-7Agⁱ was produced by P. Banks (line TC14 in a Sunstar background). The second was found in the Agrotricum Zhong 5 from China, the resistance has been located on a homoeologous group 2 chromosome by P Banks (addition line Z6). In the progeny of a F1 of the cross between Z6 and wheat, we selected ditelosomic addition lines.

Seedlings of the different genotypes were inoculated when the second leaf was half emerged. Three aphids contaminated by a PAV isolate of BYDV were deposited at the basis of each seedling. The aphids were killed five days later. ELISA test was made 15 and 30 days after inoculation.

Both sources of resistance slow down the multiplication of the virus in the plants, and have an effect on the proportion of plants which escape contamination. More than a third of TC14 plants, about 15 % of plants with Zhong 5 resistance and less than 1% of the susceptible plants escaped contamination. For the contaminated plants we observed a significantly lower multiplication of the virus (optical density) in the plants derived from Zhong 5 than in the other one. The mean optical densities 15 and 30 days after inoculation respectively were 0.2 and 0.5 for Zhong 5 derivatives, 0.6 and 0.9 for TC14 and 1.8 and 3.1 for the susceptible control Sunstar. These data suggest that the mechanism of resistance is different for these two genes

Molecular markers were found using RAPD. Five molecular markers of the gene translocated on the 7D chromosome have been found. The data obtained from a F2 of the cross TC14×Rendez-vous suggest that the fragment of the 7Agⁱ chromosome could recombine with the 7D. This has to be verified from the study of the F3. Four markers of the chromosome 2Agⁱ were found: one on the arm without any resistance gene, one close to the centromere, and two on the arm carrying resistance, one of them being probably in a distal position.

The introgression of these two systems of resistance into an agronomic background adapted to France is under progress.

In 1996, we implanted in the field a test of tolerance with plants contaminated by a PAV vectotype of BYDV and plants not contaminated protected by insecticides. We observed that the yield loss of the lines carrying the tolerance gene Bdv1, from Frontana (Anza, Frontana and an isoline of Thatcher), was lower than the one of the resistant line TC14. But all these lines were spring type and poorly adapted to our conditions, so it is difficult to choose the best system before the transfer of the resistance and tolerance genes into adapted genotypes.

Resistance to BYDVs in Chinese interspecific crosses

M. Henry,¹ M. van Ginkel¹ and Qi Shiyu²

1. CIMMYT Int., Lisboa 27, Apdo Postal 6-641, Mexico, D.F., 06600 Mexico. 2. Crop Breeding Institute, Heilongjiang Academy of Agricultural Sciences, Harbin, Heilongjiang, China

Two spring wheat lines, New LongMai 15 and New LongMai 19 (for both, n=42), were derived from crosses between Triticum intermedium and commercial Chinese bread wheat cultivars by Prof. Qi Shiyu and tested for their resistance to four BYDV serotypes at CIMMYT.

The isolates used were PAV-Mex, RPV-Mex, MAV-Mex and RMV-UK. Ten plants were inoculated with ten viruliferous aphids for each BYDV isolate; two plants were maintained free of aphids to serve as healthy checks. Bobwhite and TC14 were also inoculated and served as susceptible and resistant controls, respectively. TC14 is an Australian wheat line that also contains a T. intermedium introgression on chromosome 7D (Banks et al., 1994, Genome 38:395-405). ELISA was performed 15 and 30 days after inoculation.

Virus titers were low in the two Chinese lines and in TC14 for the four BYDV isolates. Virus titers were moderate to high in Bobwhite. Plants were assumed to be infected when the optical densities (OD) in ELISA were twice the average of the healthy checks. The results are summarized in Table 1.

Table 1: Virus titers (ELISA) measured in three presumably resistant and one susceptible bread wheat lines after artificial inoculation with BYDV.

The results indicate that the two Chinese lines are resistant to virus multiplication of the four BYDV serotypes tested. These lines have been included in the crossing activities of the Bread Wheat Program at CIMMYT in order to incorporate alien derived BYDV resistance into elite bread wheats. The resulting progenies will be made available to our collaborators. Further studies will attempt to combine this resistance and the resistance based on TC14, with tolerance genes such as Bdv1 described by Singh et al (1993, Crop Science, 33: 231-234).

Resistance to BYDV in barley

M. Henry (1) and H. Vivar (2)

(1) CIMMYT Int., Lisboa 27, Apdo Postal 6-641, 06600 Mexico D.F., Mexico. (2) * CIMMYT-ICARDA, same address.

Barley lines showing good tolerance to one or more BYDVs were screened for resistance to virus multiplication. Sixty-six lines including the susceptible and resistant checks Atlas 57 (susceptible), Atlas 68 (resistant, tolerant) were inoculated. Six plants per line were artificially inoculated with 10 aphids of the following BYDV isolates: BYDV-PAV-Mex, BYDV-RPV-Mex, BYDV-MAV-Mex. One plant was kept free of aphids and used as the healthy control. Plants were maintained in the greenhouse for 15 days. One leaf per plant was then collected to be tested by ELISA. Plants were assumed to be infected when the optical density (OD) in ELISA was more than twice that of the healthy controls.

ELISA titers obtained ranged from 0.175 to 0.909 for PAV, 0.109 to 1.375 for RPV and 0.164 to 0.886 for MAV. Three lines out of the sixty-four tested had very low ELISA titers for both PAV and MAV and high titers for RPV. These lines were:

These lines also showed tolerance in the field for the same BYDVs.

Twelve lines had low titers after inoculation with BYDV-RPV (0.111-0.249) but only three of these showed tolerance for RPV under field conditions. These three lines were:

Many of the lines tested showed reduced or no symptoms in the field, but had moderate to high ELISA titers, suggesting that tolerance was the most common mechanism.

The tested lines are high yielding barley lines from the ICARDA-CIMMYT barley program. Many of them also have multiple disease resistance. More work has been undertaken to characterize the type of BYDV resistance/tolerance present in ICARDA-CIMMYT barley program to provide suitable germsplasm for areas of South America where the virus causes severe losses to barley production.

Trial on the resistance of cereals to viruses in the South of Ukraine

L. I. Omelchenko and L. T. Babayants

Phytopathology and Enthomology Department, Plant Breeding and Genetics Institute, Oridiopolskaya doraga 3 Odessa, Ukraine

Resistance of grain crops to virus diseases has been an integral part of the research activities at the laboratory of Phytopathology and Entomology of the Plant Breeding and Genetics Institute for the last 15 years. Because of the high cost of aphid maintenance and reliability of natural BYDV infection, screening for tolerance to BYDV in wheat and barley was done under natural infection conditions.

A new field screening procedure was tested during various cropping seasons since 1996. Each genotype was planted in six 2.5 m plots. Three plots / genotype were kept free of aphids by insecticide applications and served as the healthy check for each genotype. The experiment was kept free of natural aphid infestation and pathogen infection for the rest of the season by spraying insecticides and fungicides when needed. Symptom reading were taken after flowering, using a four digits scale; the first digit for the percentage of yellowish or reddish leaf area, the second for the percentage of dwarfing, the third for the percentage of reduction in tillering, and the fourth for the harvest index. All indexes were estimated by direct comparison with the healthy check of each genotype. Three hundred genotypes of winter wheat and forty five of barley were tested under natural infection conditions. Leaf samples from selected genotypes showing BYD - like symptoms were also tested by ELISA to confirm the presence of BYDV. None of the tested genotypes were found free of BYDV. Ten percent of the wheats tested had similar tolerance to BYDV. Among winter wheat varieties, Albatros odessky, Fantazia, Krasunya, Nadiya, Odesskaya 265 could be classified as tolerant to BYDV infection. The level of tolerance of the durum wheat varieties such as Aisberg odessky, Delfin, Delta, was also high. A crossing programme was started to study the inheritance of the BYDV tolerance in the selected material.

Strains of barley yellow dwarf virus in European Russia

K. Mozhaeva, T. Kastalieva and T. Vasilieva

Group of Virology, All Russian Research Institute of Phytopathology, Bolschie Vyazemy, Moscow Region 143050, Russia

Severe epiphytotics of BYD were reported on oat and barley in many regions of the Non-Chernozem zone and in a lesser extent in the Central-Chernozem region in Russia between 1988 and 1991. A cereal sowing area of several millions hectares was infected by BYD, causing important yield losses for agriculture.

Since 1992, we study BYDV in the "All Russian Research Institute of Phytopathology". Isolation of virus, its diagnosis, disease monitoring and strain identification are our fields of investigation. In collaboration with M. Shemyakin and Yu Ovchinnicov from the Institute of Bio-organic Chemistry of the Russian Academy of Science, monoclonal antibodies and corresponding horseradish peroxidase conjugates were produced. The antigen used for mice immunization and clone selection was isolated from natural infected material (Erockina T., Kastalieva T. Zuchtungforchung, 1995, v. 1, N 2, pp. 71-74).

BYDV serotypes composition in infected samples were analyzed using these monoclonal antibodies and polyclonal antibodies from AGDIA Inc. The samples of cereals, grasses and weeds were collected in European Russia between 1992 and 1997.

Rhopalosiphum padi, Sitobion avenae, Schizaphis graminum and Metopolophium dirhodum known as vectors of RPV, PAV, MAV, SGV, and RMV were observed. Those BYDV strains were found in inspected areas.

PAV and RPV were the predominant strains in the Non-Chernozem zone. MAV and SGV strains were also found but were less frequent. Besides these four serotypes, RMV was observed in Povolzhskiy region. PAV, MAV and SGV were detected in the Central-Chernozem region.

In 1993-1997, an evaluation of BYDV incidence was conducted in cereals crops from Moskovskaya Oblast (Central region). In 1994, detail studies were done in oat and barley plots at the Research Institute of Agriculture in the Central region of the non-Cernozem zone. The incidence of infection was assessed on oat and barley using visual observations and sample analysis by ELISA. Incidence in barley was not high. It fluctuated amongst plots between 3,3% to 8,6% according to visual evaluation and between 2,7 to 7,9% according to ELISA analysis. RPV was predominant with approximately 70% of the infected barley samples, while PAV was found in 40% of the samples. Approximately one third of the samples were infected by both PAV and RPV. Higher levels of infection were recorded in oats. Visual inspection suggested that 34% of oat samples were infected, however, only 19% of the samples reacted positively in ELISA. The predominant BYDV serotype was PAV (68%), followed by RPV (26%). 22% of the infected oats had a mixture of PAV and RPV [K. Mozhaeva et al., Selekziya I Semenovodstvo, 1:21-24 (1997)].

Table 1. BYDV occurrence in several Russian regions

* - Only RPV and PAV were detected.

In the main European Russia, aphid-vectors of BYDV are present and there is always sources for primary virus spread to small grain crops (winter crops, grasses and weeds), suggesting that BYDV could be a serious problem in this region of the world. Serotypes composition of BYDV and the degree of cereal disease are dependent upon the aphid species present and indirectly upon the weather conditions influencing the life cycle and the spread of the aphids.

BYDV incidence and epidemiology of wheat in Shaanxi Province of China

Y.F. Wu and N.S. Wei

Department of protection, Northwestern Agricultural University, Yangling, Shaanxi 712100, China

Wheat is an important grain crop in northern China. Yellow dwarf disease caused by barley yellow dwarf luteovirus (BYDV) is a very important wheat disease, causing severe yield losses in 1966, 1970, 1973, 1978 and 1980 in Shaanxi. Since 1980, this disease occurs every year and has extended quickly to neighboring wheat regions. Preliminary results of aphid transmission experiments and enzyme linked immunosorbent assays (ELISA) indicated that GPV is the most frequent serotype, but PAV as well as mixtures are also found frequently on winter wheat. GPV and DAV are the predominant BYDV types in Shaanxi. In natural conditions, infection of wheat seedlings by GPV and DAV depends on the presence of different aphids vector species. GPV is usually transmitted by Schizaphis graminum and Rhopalosiphum padi, and DAV by Acyrthosiphum dirhodum and Macrosiphum avenae. GPV and DAV are the Chinese BYDV serotypes that present no reaction with antiserum to PAV, MAV, SGV, RPV and RMV.

Many years of observation showed that climatic variables are important epidemiological factors. If air temperatures remain relatively low in June, become higher in October or warmer during autumn, then increase quickly in March of the following year, in conjunction with drought, barley yellow dwarf disease occurs heavily. Possible explanations to this relation between BYDV incidence and epidemiology, are the air temperature that correlates with fluctuations of aphid population (number and time of appearance) in early spring, when aphid vectors, in particular S. graminum transmit GPV from weeds to wheat seedlings. Population density of S. graminum in March may be suggested as an indicator to forecast BYDV incidence and epidemiology. Weeds and over-wintering infected hosts as a natural reservoirs, such as Avena fatua, Sorghum halepense, Stipa brachychaeta and Cynodin dactylon play an important role in BYDV epidemiology.

BYDV acquisition and inoculation are associated with the occurrence and duration of characteristic stylet activities during phloem penetration by aphids

G. Powell and J. Hardie

Department of Biology, Imperial College at Silwood Park, Ascot, SL5 7PY, UK

Phloem-restricted luteoviruses are entirely dependent upon aphid vectors for transmission; they are acquired when the insects ingest phloem sap, and inoculated when they inject saliva into plants. Stylet penetration activities by aphids can be studied by making the insect and plant part of a DC electrical circuit to generate sequences of characteristic waveforms called electrical penetration graphs (EPGs) [Tjallingii, Ent. Exp. Appl. 24:731-737 (1978)]. We have used this technique to investigate the aphid stylet activities which are associated with acquisition and inoculation of an MAV-like isolate of BYDV by the grain aphid, Sitobion avenae, and an RPV-like isolate by the bird cherry-oat aphid, Rhopalosiphum padi. Wingless adult aphids were allowed up to 6h electrically-recorded access to a BYDV-infected oat plant, and then transferred to a test seedling for a 2 day period to allow for vector latency. Insects were subsequently transferred to a second test plant for electrical recording of stylet activities during a range of inoculation access periods. This was followed by transfer to a third test plant, where the aphids remained for a 4 day period. All test plants were assayed for BYDV infection using ELISA 4-6 weeks following aphid access.

Periods of intracellular stylet tip location are registered as potential drops during electrical recording; these may be short (all cell types) or sustained (phloem sieve elements only). Two waveforms occur during extended phloem sieve element penetration by aphid stylets; firstly E1, which may later change to E2 (E1+E2) e.g. [Prado & Tjallingii, Ent. Exp. Appl. 72:157-165 (1994)]. Most aphids (98%) which subsequently transmitted either virus to one or more test plants showed E1+E2 during penetration of the infected plant. Separate analysis of E1 and E2 durations suggested that E2 is associated with virus acquisition, as insects which transmitted to test plants showed longer total E2 occurrence on the source plant than non-transmitting aphids. Most aphids (86%) which transmitted to the test plant during recorded inoculation access showed either E1 alone or E1+E2. Inoculation tended to coincide with longer periods of E1. However, some insects which transmitted virus showed E1 for <5min, and 14% of inoculations were effected by aphids that showed no E1/E2, indicating that transient phloem punctures occur which are indistinguishable from penetration of other cell types using EPG recording.

The results suggest that electrically-recorded transmission characteristics of BYDV-MAV (BYDV subgroup 1) and RPV (subgroup 2) are very similar: acquisition of both viruses by aphids requires sustained periods of phloem contact, but inoculation can occur during very brief punctures of phloem tissue. These findings are in agreement with a previous study, where transmission of BYDV-PAV (subgroup 1) was investigated [Prado & Tjallingii, Ent. Exp. Appl. 72:157-165 (1994)].

Interactions between BYDV and a parasitoid in a shared aphid vector/host

Petra Christiansen-Weniger, Glen Powell and Jim Hardie

Department of Biology, Imperial College at Silwood Park, Ascot, SL5 7PY, UK

The pest status of cereal aphids may be reduced by parasitoid wasps whose larvae develop inside their insect hosts. These natural enemies are being increasingly looked to and implemented in modern aphid control strategies, but interactions with virus transmission have so far received little attention. We investigated interactions between an MAV-like isolate of BYDV and the aphid parasitoid Aphidius ervi (Hymenoptera: Aphidiidae) in the shared aphid vector/host Sitobion avenae with surprising results [Christiansen-Weniger et al., 86:205-213 1998)].

The impact of virus presence became immediately apparent when observing the mortality of aphids that had been parasitised by A. ervi. Significantly more insects died before formation of the aphid mummy (the parasitoid pupal cocoon inside the dead aphid) when both virus and parasitoid were present than when the parasitoid was present without virus. In addition, aphid dissection revealed that parasitoid development was significantly delayed in the presence of virus (on the 14th day after oviposition 95% of A. ervi developing in virus-free aphids had reached the pupal stage, whereas only 39% of those developing in hosts carrying virus were pupae). The virus moves through the haemolymph from the gut to the salivary glands and may therefore interact with the developing parasitoid, but the physiological mechanisms responsible for the increased aphid mortality are unknown. However, the results indicate a low suitability of viruliferous S. avenae as hosts for A. ervi. Studies were then made on oviposition success in virus-free and viruliferous aphids, to determine whether host suitability could be recognized by the ovipositing parasitoid. Indeed, female A. ervi appeared to discriminate, with significantly more eggs being deposited in virus-free aphids (56%) than in viruliferous aphids (39%). But does the presence of the parasitoid affect transmission of the virus?. A daily transfer of parasitised aphids to virus-free host plants after the initial virus acquisition access period showed that virus transmission was not reduced by the presence of the parasitoid developing inside the aphid, at least up to a point close to host death and mummy formation.

Thus, while the transmission of BYDV by the aphid vector remained unchanged by parasitisation, parasitoids may selectively avoid oviposition in viruliferous aphids as retarded development and/or premature death may result. Such observations indicate that the success of aphid parasitoids as control agents for BYDV in the field may be restricted.

© CIMMYT December 1998

Barley Yellow Dwarf Newsletter No. 7