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Diseases and pests in energy crop plantations
0961-9534192 $5.00 + 0.00 8 1992 Pecgamoa Press I&l
Biomaw ad Bioenergy Vol. 2, NW l-6. pp. &X4,1992
Printed in Great Britain. All rights nserved
DISEASES AND PESTS IN ENERGY CROP PLANTATIONS D.
J. ROYLE*andM. HUBBEst
* University of Bristol, Department of Agricultural Sciemes, AFRC Institute of Arable Crops Research, Long Ashton Research Station, Bristol BS18 9AF, U.K. t Faculty of Forestry, University of Toronto, Ontario, Canada M5S 3B3
ABSTRACT The Pest and Disease Management Activity was established in response to an awareness of the potential importance of pests and diseases in woody biomass production systems. Ammal surveys in Canada, Eire, England, N. Ireland, Scotland and Sweden from 1987-91 confirmed that rust disease (Melampsoru spp.) is currently the most serious problem in willow biomass production in Europe. Other pests and diseases cause problems only on a local scale, though they could become more generally severe. Increasing clonal susceptibility to rust and an an increasing association of premature defoliation with lower rust severity levels have occutred over the period. A high degree of intrinsic variability in pathogen populations is expressed as fourfonnue specides (Espp.) and at least eight pathotypes of the most common rust species, M. epiteu. The same Espp. appear to occur among pathotypes in Sweden, New Zealand and the UK. A network of field experiments has been established, in collaboration with the Joint Trials Activity, to enable the rust pathotype composition to be compared between five countries. It also aims to identify an international set of standard willow clones to be utilized for characterizingrust pathotypes globally. KEYWORDS Diseases, Pests, Biomass crops, Energy crops, Willow, Poplar, Rust diseases, Melumpsoru, Septoriu, Chrysomelids, Biotechnology, Disease assessment INTRODUCTION The Pest and Disease Management Activity was established in 1989 in recognition of increasing problems that pests and diseases were beginning to cause within certain IEA Bioenergy Agreement countries in the production of woody biomass crops for energy. At this time there were many reports of investigations on pests and diseases of poplar, though not perhaps specifically in relation to energy production. Much was already known therefore about their identity and the problems they were likely to cause. In contrast, little was known about disorders of willows. Consequently, within the earlier IEABA Task II programme (1986-89),surveys had been initiated by the Willow Breeding Activity to identify the diseases and pests occurring in short-rotation willow coppice plantations and to make assessments of their importance (Hunter et al., 1988, 1989). Development of the surveys was considered necessary as a framework for the new Activity, in order that changes in the prevalence and severity of pests and diseases could be determined in relation to clone and site factors regionally, nationally and internationally. Initially, the surveys had identified the crucial importance of rust (Melampsoru spp.) as threatening the establishment and productivity of willow energy crops within the UK and Sweden, and work in the 45
46
D. J. ROYLE and M. HUBBES
Task V Activity began to analyze the rust population structure for variability with respect to different sites and the responses of different clones. Problems of willow rust are shared by Canada, Sweden, Eire, N. lreland, Scotland and England, and they have tended to dominate the work of the Pest and Disease Management Activity. This has led to studies on rust epidemiology, the scales of yield loss caused by the disease and in developing standard methods of rust assessment which would be suitable in both research and breeding programmes. There has been a close co-operation with the Joint Trials and Exchange of Genetic Material Activities with whom the Pest and Disease Activity has enjoyed two fruitful joint workshops, in Belgium 1990 and in Iowa 1991. Both of these Activities have themselves interests in pests and diseases, as factors in clonal performance in field trials and in relation to the development of resistance in breeding progmmmes. Within a separate section of the Activity, co-operative work on resistance mechanisms and biotechnology has been concerned with the development of tissue culture, isozyme and molecular and biochemical techniques for studies on Septoria musiva and bacterial canker of poplar and Melampsora rusts of poplar and willow. This report presents an outline of selected aspects of the Activity progmmme. INTERNATIONALSURVEYSOF WILLOWPESTS AND DISEASES, 1987.91
With the co-operation of observers in Canada, Eire, England, N. Ireland, Scotland and Sweden, annual surveys were done over 5 years on clonal willow plantations and plots (Table 1).
Table 1. Number of clone plantings assessed during surveys, 1987-91 -~__~~-__~~-_1_~~~~_~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~-~~~~~~~~~~~~~~~~ N. Ireland Scotland Eire Sweden Year England Canada 1987 13 93 5 94 25 1988 27 29 121 32 32 43 1989 30 39 141 36 80 30 1990 37 41 163 16 92 61 1991 27 37 176 16 _ _
Pests and diseases were assessed according to a simple questionnaire (Hunter et al., 1988, 1989, 1991) which was developed at Long Ashton Research Station where the surveys were co-ordinated. Recording was made simple to facilitate comparison of results from different observers, and severity classes assigned to different disorders were broad to emphasise extremes and to encourage standardizationin reporting. Symptom types were in 6 groups: (i) leaf rust, (ii) leaf and shoot tip dieback, (iii) disorders and cankers of woody stems, (iv) defoliation, (v) stool death, (vi) other/insect damage. Leaf infection due to rust was judged over an entire clonal planting as: 0 = no rust; 1 = low, occasional leaves infected; 2 = moderate, frequent leaves infected; 3 = severe, many leaves infected, rust pustules often in dense aggregations. Defoliation attributable to rust (potential yield loss) was scored as an estimate of the proportion of shoot lengths affected. Identification of disorders involving leaf and shoot-tip die-back could be difficult but was encouraged by listing various possible causes. As for leaf rust, stem disorders were gmded in 4 severity levels. Participants were asked to record possible causes of stool death, if known. Opportunity was given for individual comments on other, non-specific disorders, and on the general state of the plantation under scrutiny. As the surveys progressed, the relative incidence of feeding damage due to insects became apparent and, in the development of a computerized database, it was necessary to categorize beetle damage in a similar way to that for rust.
Diseasesand pests
47
Two assessments were done each year, in July/August and in September/October. Wherever possible, the same clone plantings were monitored over the 5 years. Whilst it was hoped that the questionnaire would be adopted by all patticipants, the observers in Canada and Sweden resorted to altemative ways of communicating their observations, mainly because of time pressums, though they were closely guided by the questionnaire.
At UK sites problems due to tbngal pathogens other than rust were rarely recorded, but in Canadian and Swedish plantings damage due to willow scab, Fusicludium saliciperdum, and branch cankers caused by Glomerella miyabeana, featured in older plantations. Damage by both these pathogens occur& mainly as shoot tip die-back and cankering of rods. In Canada, a common disorder on Sulix capreu, S. fragilis and S. alba hybrids was Marssoninasalicicola, attacking shoots and branches and causing leaf spotting. However, Fusicludium was the most serious problem, especially on S. lucida clones. At one site, Maple, Ontario, in 1990, 10/l 1 willow types were infected by this pathogen, only S. exigua remaining healthy. Many S. matsudunax S. alba crosses were also severely damaged, often with crown die-back. Swedish plantations also suffered from Marssonina leaf spotting, on S. caprea and S. fragilis type crosses. A die-back problem, otten associated with S. viminalisclones 78021, 78112 and 78183 and with S. daphnoides 79149, was identified in 1991 as associated with ice nucleation activity of the bacterium Pseudomonassyringue. This has become of increasing importance and is now serious with die-back progressing into rootstocks and causing stool death.
The most widespread damage from insects over the survey period was caused by Chrysomelid leaf beetles, e.g. Phyllodectavulgatissima. In the UK an average of 42% of plantings were affected, 8% severely so. Other locally damaging pests on mature stands included the willow weevil, Cryporrhynchus Zupathi,especially in Canada during the cool, wet summer of 1990. In 1991, damage by the potato leaf hopper, Empoasca fabue, was reported for the first time in Canada. In Sweden damage due to Terminalis midge on S. viminafisclones was consistently recorded and it also occurred to some degree in most UK plantations. The plant sucker, Aphrophoru sulicis, occurred in 1987 and 1988 on many S. butjuticu clones in the UK though it has not since been damaging. Eryophyid mite damage has also occurred on the same clones in UK plantings and on a number of clones in Sweden in 1991. Stem aphids, Melunoxantherumsalicis and Pterochforussp., were seen on many UK plantings in 1989and 1990, but not since.
Even though its intensity has varied between years and clones, rust has consistently been the most widespread, severe disorder in coppice willow plantations, especially in the UK and Sweden. Defoliation caused by rust has occurred frequently at many sites and on many clones and has almost certainly been responsible for heavy losses in biomass yields and progressive decline in the vigour of many plantings. UK In England rust incidence and severity was greatest in 1988 when more than 92% of clone plantings were infected, and over 60% had severe early defoliation. Most of this occurmd on S. bwjuticu clones, especially Korso which because of its extreme susceptibility is no longer regarded as of potential commercial value. Rust and associated defoliation has remained severe in many clonal plantations in recent years.
48
D. J. ROYLE and M. HUBBES
Even though there has been a gradual decline in the proportion of N. Ireland plantings afTected by severe rust and an increase in that affected by light attacks since 1988, defoliation has nevertheless increased and has thus appeared to be occurring at progressively lower rust levels. Over the entire survey period only 16% of clone plantings remained without rust, most of them (67%) the clone SQ83 (S. viminalis x triandra), which has so far remained resistant. In Scotland the frequency of rust early in the season increased after 1989, though at low intensities. Final seasonal severity and defoliation increased over the same period. This is reflected by an increase in rust on S viminalis clones in Scotland and elsewhere in the UK over the years. The data from UK plantings overall early in the season indicated trends towards an increasing proportion with moderate-severe rust over the 5 years. This is consistent with the disease appearing earlier or else developing at a progressively faster rate within plantings. These trends were not evident at the end of the season, however, the proportion without rust (13-20%) remaining fairly constant after 1988. Around 50% of plantings showed early defoliation in each of the last 4 years. In general, defoliation of S. viminalis clones occurred at lower rust severities than S. butjatica types. No cases of rust-induced defoliation were reported on 5. viminalis clones in 1987 but since then early rust attacks have led to defoliation. More early defoliation occurred on both S. viminahs and S. burjatica clones in 1988 than in other years, with 56% and 76% of plantings affected, respectively. Most records of rust in the surveys were of leaf infections. However, stem cankering caused by a new form of the rust pathogen has been increasing. The first records in 1988 were in only 8 plantings but, by 1991, 27/240 contained rust cankers. Overall, the S. viminalis clones, especially Bowles Hybrid (2 1.6% of plantings with cankers) were most severely affected. In 1991, several other clones were also affected, including the S viminalis clones Campbell, English Rod, Mullatin and Gigantea, and the hybrids S. x sericans, S. x hirtei, S. x dasyclados, S. x rubra and S. x calodendron.
Sweden. With the largest area of any country planted to biomass willows, Sweden has not repotted serious rust epidemics in recent years. In 1987 rust was most common on S. burjatica clones; exceptionally there were few problems on S viminalis types. As in the UK, many more leaf rust pustules had to occur on S. viminalis than on S. burjatica clones before senescence and leaf fall occurred. Less rust was recorded generally in 1988 and 1989 with infections occurring only late in the season. Even S. burjatica clones 056 and 075, which were infected severely before, were free of early rust at most sites. In 1990 there was again little rust except on S. viminalis 683 and some 15 S. caprea clones at Uppsala, and in some plantings in the south where other S. viminalis clones were severely attacked. There were few problems again in 199 1.
Canada. There has been a steady increase in the number of willow types with serious rust infections in Canada. In 1987 severe rust with defoliation did not occur until October and then at only one site on S. amygdaloides and S. pellita. This was also the case the following year with S. amygdaloides clones the worst affected and S. exigua x petiolaris and x eriocephala showing symptoms by September. Severe rust was detected earlier in 1989 with some S. amygdaloides clones defoliating by August at Maple, Ontario although many S. pellita and S. lucida clones were without tust. At two other sites only S. Iucida and S. discolor clones showed no rust throughout the season when it occurred on S. viminalis xpurpurea but not on the x triandra nor x caprea hybrids. Here several S. purpurea clones which were moderately infected in 1988 remained without rust in 1989. The same three sites were assessed in 1990 and 1991 but only at Maple late in the season was rust severe on two clones of S. amydaloides and two clones of S. exigua in 1990, and moderate on S. eriocephala and S. petiolaris in 1991. In general, rust in Canada is not as severe as in other countries and only relatively few clone types seem to be susceptible. Clone types originating from Europe and which were recorded as severe in the UK and Sweden, failed to exhibit high levels of rust and only 3/25 had any rust at all.
49
Diseases andpests
RUST VARIABILITYAND CLONAL RESPONSE Results from the surveys have indicated that perhaps the most difficult obstacle to the successful control of willow rust is the extreme variability of the pathogen The evidence suggests that variability is expressed at various levels: in the response of different clones, within and between different plantings of the same clone, and between different regions and countries. Within the Activity we set out to assess this variability in the UK, Canada and Sweden and to develop a standard set of differential clones on which forms of rust can be characterised.
Pathogenicity studies at Long Ashton, mainly using an in vitro inoculated leaf disc method, have shown that in the UK there are at least three species of Mehzmpsoru occurring on biomass willows: M. epitea var. epitea, the most widespread and common, M. caprearum and M. rib&i-viminalis. Furthermore, within the complex species M. epiteu var. epifea, five “form species (Espp.)” can be recognised within which there are at least eight “pathotypes”or “races”. These can be distinguished only by their reactions on differential willow clones (Table 2). The Esp. larici-epitea typica is represented by the 3 pathotypes LET1 (mainly infecting S. viminalis clones), LET2 (from the hybrid gcupreu x cinerea x viminalis 445 de Biardii) and LET3 from S. x culodendron. The Esp. larici-retusue is represented by the two pathotypes LRl, which causes the outbreaks on S. burjatica Korso, and LR2 from S. himalayas. The Esp. larici-daphnoides is represented by LDl from S. duphnoides Rubenima, and the Esp. ribesii-pwpureu by RP1 from S. purpurea Uralensis. Finally, the new “shoot-infecting”Esp. of rust from S. viminulis Bowles Hybrid occurs as a single pathotype, SIF. Table 2.
Proposed differential willow hosts and their reactions to pathotypes of Melampsora epitea var. epitea
Differential host viminalis ‘Mullatin x boydii x stipularis x calodendron butjatica ‘Korso’ himalayas daphnoides ‘Meikle’ purpureu ‘Jagellonica’
Pathotype LET1 LET2 + _ +
LET3
SIF -
LRl
LR2
LDI -
RPl
+ +
+ _ +
+ -
+
+
+
+
+
+
-
-
-
-
+
-
-
_
-
-
_
-
-
-
_
+
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+
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_
+ -
+
Since Suk species are either naturally distributed or introduced worldwide, it is perhaps not surprising that h4. epitea has been reported in Eurasia, N. America and recently Australia Exchange of information within the Pest and Disease Activity has suggested that in Sweden (Rams@, 1989) the rust pathogen shows five distinct patterns of pathogenic@ when tested with S. &phnoides “ESO”,S. viminalis x caprea, and several clones of both S. viminalis and S. x dapyckrdo (The name S. x dusycludos is used on the continent to refer to two forms called in the UK S. bmjutica Nasarov (synonym S aquatica grundis, see Bean (1980)) and S. x &sycluui~ Wimm. (Stott, 1991)). The Swedish pathotypes seem to belong to the form species larici-epitea typica, laricidaphnoiaks and possibly Zarici-retusae. In New Zealand, rust infection was observed for the Erst time on S. incuna x duphnoides in 1978. This was a new rust apparently similar to M. epiteu which caused severe attacks on S. viminalis and hybrids in 1985 (Spiers and Hathaway, 1988). From the host lists in New Zealand, the iU. epitea rusts appear to belong to larici-daphnoiaks and larcici-epitea @pica.
50
D. J. ROYLE and M. HUBBES
Since it could be dangerous to exchange isolates of the airborne rust pathogen between countries for pathogenicity testing, a network of five field trials was established in March 1991, in collaboration with the Joint Trials Activity, at Long Ashton, Loughgall, N. Ireland and Craibstone, Aberdeen, Uppsala, Sweden and Ontario, Canada. Each trial incorporates small (5 x 5 stool) plots of 24 clones, (12 from UK, 6 from Canada and 6 from Sweden), replicated five times and selected according to a range of responses to rust in each country. The project aims to compare rust populations in order to determine which pathotypes occur in the different countries. It also aims to establish the suitability of the clones for studying pathotype x environment interactions. From this we hope to develop an international set of standard willow clones for characterizing rust pathotypes globally. In the establishment year, data has already indicated a different spectrum of rust pathotypes between the countries. DISEASE ASSESSMENT Field research on biomass diseases, the IEA rust/clone trials in particular, demands standardized methods for monitoring disease levels within plantings. Also, there will be an increasing need for monitoring as interest in growing amble energy crops increases. The underlying reason for this monitoring will govern the scale, frequency and required accuracy of the assessments that are made. However, assessments that are unrepresentative of the planting are worthless, whatever the reason for the assessment. The reliability of any attempt to quantify disease is strongly influenced by the protocol used to obtain the samples, and the accuracy and precision (defined by Campbell and Madden, 1991) of the visual disease severity assessments. These factors have been investigated in a 6-month progmmme at Long Ashton, within the IEA Activity, and utilizing the co-operation of participants in the rust/clone trials. The study focused on willow rust because of the priority need to develop standarized methods of assessing this disease.
The spatial and temporal spread of rust was investigated in a small plot (12 x 19 stools) of S. dasyc1udos Wimm. A single shoot was randomly selected for every stool, and rust incidence and pustule frequency on every 20th leaf were recorded weekly. Preliminary analysis using mapping suggested that the disease spread was not focal. However, further analyses will be required to confirm this and more work is needed if recommendations about sampling are to be provided with confidence.
It is widely accepted that disease assessment keys can improve the reliability of visual estimates of disease severity, so it perhaps follows that they are essential to good assessment. A simple key for willow rust assessment was produced by McCrackenand Dawson (1992). However, currently, no key has gained wide acceptance and so the first aim of this study was to discover whether any key was necessary. Method. A large number of leaves of S. burjuzicuKorso were collected from the IEA rust/clone trial at Long Ashton. Of these, 30 were selected which provided a range of rust severity typical of that found in the field. Professional photographs were produced of the abaxial surtbces of the leaves, labelled and placed singly in transparent cellophane sleeves. A set of photographs, with score sheets and instructions, was mailed to each of the other four groups participating in the rust/clone trials. Observers were instructed to use a hand lens (x10) when making an assessment. There were several
Diseases and pests
51
observers from each group, including Long Ashton, who participated. Objective estimates of disease severity were obtained using video image analysis. Objective and visual estimates of disease severity were logit transformed to stabilize variance. Least squares regression models were obtained for each observer for visual fitted against objective estimates. The similarity of these models was compared in the way described by Parker (1991). Results. Even observers from the same research group were found to differ greatly in their perceptions of disease severity (p>O.UZ). For most observers the errors in assessment were attributable to gross over-estimation of severity. This problem has been reported for visual assessments of brown tust of barley (Puccinia hordei) by Beresford and Royle (1991). Over-estimationwas a particular feature of the assessments of observers who tended to ascribe severity into bands rather than on a linear scale. It is suspected that such individuals generally made assessments with reference to disease scales. The evidence obtained indicated that assessment into bands should be discouraged because it was apparent that even gross differences in disease intensity could be missed. A key previously untested (Parker, 1991) was refined to correspond to the shape and size of willow leaves and tested for its efficacy in improving the reliability of assessments. The key is based on a generic leaf grid made-up of 1% sectors, The coordinates of this grid were incorporated into a computer programme that includes a scaling feature so that leaf dimensions (length & breadth) can be inputted allowing any size of leaf to be generated. Three such leaf grids printed on acetate and covering the range in leaf size of the photographs were provided to the same groups for a second assessment. Observers were instructed to use this key as an overlay, Estimates provided by different observers were, in general, more similar in the second assessment than previously achieved (Fig. 1). Moreover, vast over-estimation was eliminated, so the assessments were more accurate. The effect on assessment precision was less clear. For several observers there was an obvious improvement, but for others improvement was either marginal or, in a few cases, precision was slightly reduced. Further work is necessary to clarify the effect of using the key on assessment precision. Several observers indicated that they found the use of the overlayed key either difficult or too time consuming. We propose that the key should be printed onto paper and used as a guide at the start, and subsequently as necessary, for each assessment. It would be prudent to undertake further investigation to ensure that the key is effective when used in this way. RESISTANCEMECHANISMSAND BIOTECHNOLOGY This part of the Activity programme aimed to develop tissue culture and in vitro techniques for Sepzotiu m&vu and bacterial canker on poplar and Melampsoru spp. on poplar and willow. It also compared pathogen isolates using isozyme and other biochemical methods and investigated resistance mechanisms by molecular and biochemical techniques. Septoriu musivu infects native and hybrid poplars throughout N. America. Originally it was considered to be endemic in Populus bulsuminrjkra,stands but when hybrid poplars were used in plantings it developed into a lethal canker disease on many clones. In the mid-1970’shybrid poplar plantations in the north central USA were badly damaged by S. musivu (Ostry and McNabb, 1985). Hybrid poplars are expected to play an important role in future energy plantations and the impact of Seproriu canker must be determined and control methods developed (Hubbes and Wang, 1990). Cooperation between scientists in Belgium, Canada and the USA within progmmmes of this and the Exchange of Genetic Material Activities has resulted in significant progress being made towards this goal.
52
D. J. ROYL!?and M. HKJBBES
Since S. musivu does not occur in Europe, genetic poplar material is being exchanged to test its field response to the pathogen (Strobl er al., 1990, Mottet and Vallee, 1990). Simultaneous inoculations in Quebec and Ontario of 150 clones from Belgium, with two Seproriu strains, are also being done. Several clones with strong resistance to the disease have been found. Tissue culture and new molecular biotechnology techniques offer many advantages for poplar improvements over field screening tests (Ostry, 1991). The discovery that somatic variation can be induced and recovered by the passage of plant cells through a tissue culture cycle has opened up a new field of investigation. A sensitive in vitro bioassay was developed for assessing the resistance of poplar clones to Sepzoriu leaf spot (Ostry et al., 1988). The results correlated well with those from previous field trials. A recent study involved the occurrence and extent of phenotypic variation in Melumpsoru rust resistance in Pop&s deltoides somaclones generated from leaf callus. A race-specific interaction was found among regenerates for the latent period and uredia per unit leaf area (Ostry, 1991). The importance of the magnitude of variability in pathogenicity within a pathogen population has already been noted, in regard to willow rust. This is also important in developing resistance through somaclonal variation and in screening for resistance in conventional breeding programmes. S. m&vu isolates have been grouped by isolation of the pathogen’s mitochondrial, ribosomal and nuclear DNA (Hubbes and Wang, 1990). According to these authors, RFLP’s and DNA fingerprinting give best results, showing complete homology between several isolates. Strains that are homologous in their fingerprint are identical, regardless of the clonal host from which they are derived. No linkages have been found between the RFLP banding pattern and strainal virulence. Expansion of this work is planned, with larger samples, since isoxyme analysis has identified some isolates with a different banding pattern (Ostry, 1991). Another approach to develop disease resistant material is to engineer “subtle resistance genes”, such as proteinase inhibitors, into the target plant. McNabb et al. (1990) hypothesize that using a gene that codes for the production of a proteinase inhibitor would generate the stable resistance desired for insect and pathogen management strategies. Several poplar hybrids have been transformed with Agrobucterium fumefuciens strain A281 via four binary vector plasmids (McNabb et uZ., 1990). To date, 200 different putative transformed clones carrying components of the proteinase inhibitor II gene have been produced. One component, the wound inductive promoter, was used with the reporter CAT gene. Systemic promoter mRNA has shown the promise of using wound-inductive promoton as regulators for other resistance gene constructs. It is hoped that the proteinase inhibitor is also active against the proteinases of S. musivu. Bioassays showed encouraging results (McNabb ez al., 1990).
ACKNOWLEDGEMENTS The work of the Pest and Disease Management Activity has been possible only with the considerable contribution and support from a number of people. In particular, we are grateful to Mr.T. Hunter for co-ordinating the surveys, Dr. M.H. Pei for the pathogenic@ testing and Dr. S.R. Parker for supervising the disease assessment work We also thank all those who co-operated in collecting data for the surveys and in the disease assessment exercise, especially B. Beatson, M. BuIfIn, M. Dawson, J. Ford-Robertson and M. Ramstedt. Funding by the Energy Technical Support Unit for the Department of Energy for the UK rust/clone trials and for other aspects of the research at Long Ashton is gratefully acknowledged.
REFERENCES Bean, W.J. (1980). Trees and Shrubs Hardy in the British Isles. VoLIV. John Murray, London Beresford, R.M. and D.J. Royle (1991). The assessment of infectious disease for brown rust (Puccinia hordei) of barley. Plant Path., 4Q, 374-38 1.
Diseasesand pests
Campbell, C.L. and L.V. Madden (1990). Introduction to Plant Disease EpidemioZogv John Wiley and Sons Inc., USA. Hubbes, M. and CL. Wang (1991). Strainal identification of Septoria musiva. In: Proceedings of the Joint Meeting of the IEA Task 5 Activity Groups on Exchange of Genetic Material, Joint Triab of Aldus, pormlus and &li~ and Pest and Disease Management, 1990, Geraardsbergen, Belgium, ~~43-56. Hunter, T., D.J. Royle and K.G. Stott (1988). Diseases of Saliwbiomass plantations: results of an international survey in 1987. In: Proceedings of the IEA/BA Task 2 Workshop Biotechnology Development, 1987, Uppsala, Sweden, pp.37-48.
Hunter, T., D.J. Royle and K.G. Stott (1989). Diseases of Salti biomass plantations: results of an international survey in 1988. In: Proceedings of the ZEA/BA Task 2 Workshop WiZlow Breeding and Biotechnology Development, 1988, Long Ashton, Bristol, UK, pp.68-75.
Hunter, T. and D.J. Royle (1991). Diseases and pests of Saliwenergy plantations: international survey results in 1989 and 1990. In: Proceedings of the Joint Meeting of the IEA Task 5 Activity Groups on Exchange of Genetic Material, Joint Trials of k pomtlus and && and Pest and Disease Management, 1990, Geraardsbergen, Belgium, pp.27-3 1. McCracken, A. and M. Dawson (199 1). Clonal response in Salix to Melampsora rusts in short rotation coppice plantations. Eur. J. For.(in press).
McNabb Jr., H.S., N.B. Klopfenstein, S.A. Heuchlin, and R.W. Thornburg (1990). The integration of genetic engineering into Iowa State University’s woody biomass research program. In: Proceedings of the Joint Meeting of the IEA Task 5 Activity Groups on Exchange of Genetic Material, Joint Trials of A.&s,, poDulus and && and Pest and Disease Management, 1990, Geraardsbergen, Belgium, pp.66-77. Mottet, M.J. and G. Vallee (1990). Belgium poplar exchanged with Quebec, Canada. In: Proceedings of the Joint Meeting of the IEA Task 5 Activity Groups on Exchange of Genetic Material, Joint Trials of & Populus and &_& and Pest and Disease Management, 1990, Geraardsbergen, Belgium, pp.97-103. Ostry, M.E. (1991). Somatic variation in disease resistance of poplar. In: Proceedings of the Joint Meeting of the IEA Task 5 Activity Groups on Exchange of Genetic Material, Joint Trials of Alnus, Popuius and Salix and Pest and Disease Management, 1991, Iowa, USA. In press. Ostry, M.E., R.E. McRoberts, K.T. Ward and R. Resendez (1988). Screening hybrid poplars in vitro for resistance to leaf spot caused by Septoria musiva. Plant Dis., l&497499. Ostry, M.E. and H.S. McNabb Jr., (1985). Susceptibility of Populus species and hybrids to disease in the north central United States. Plant Dis., @, 755-757. Parker, S.R. (199 1). Studies on some factors influencing the reliability of disease measurements in winter wheat crops. Ph.D. thesis, University of Bristol, UK. Ramstedt, M. (1989). Identification and characterization of pathogens important in Swedish Salix plantations. In: Proceedings of the ZEA/BA Task 2 Workshop WillowBreeding and Biotechnology Development, 1988, Long Ashton, Bristol, UK, pp. 82-87. Spiers, A.G. and R.L. Hathaway (1987). Melampsora rusts of Salt&in New Zealand. In: Proceedings of the IEA/BA Task 2 Workshop Biotechnology Development, 1987, Uppsala, Sweden, pp.6569. Stott, K.G. (199 1). Nomenclature of the promising biomass coppice willows, Salix x sericans Tausch ex Kern., Salix dasyclados Wimm. and Salix “Aquatica Gigantea”. Bot, J Scotl., 46, 137-144.
Strobl, S., K. Fraser and R. Tavares (1990). Belgium poplar exchange with Ontario, Canada In: Proceedings of the Joint Meeting of the IEA Task 5 Activity Groups on Exchange of Genetic Material, Joint Trials of Alnus, Populus and Salix and Pest and Disease Management, 1990, Geraardsbergen, Belgium, pp. 104- 113.
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D. J. ROYLE and M. HIJBBES
54
6
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Fig. 1. Comparison of the assessments made by three observers from one site collaborating in the IEA nut/clone interaction experiments (a) without the new key, and (b) with the new key as an aid.
Biomaw ad Bioenergy Vol. 2, NW l-6. pp. &X4,1992
Printed in Great Britain. All rights nserved
DISEASES AND PESTS IN ENERGY CROP PLANTATIONS D.
J. ROYLE*andM. HUBBEst
* University of Bristol, Department of Agricultural Sciemes, AFRC Institute of Arable Crops Research, Long Ashton Research Station, Bristol BS18 9AF, U.K. t Faculty of Forestry, University of Toronto, Ontario, Canada M5S 3B3
ABSTRACT The Pest and Disease Management Activity was established in response to an awareness of the potential importance of pests and diseases in woody biomass production systems. Ammal surveys in Canada, Eire, England, N. Ireland, Scotland and Sweden from 1987-91 confirmed that rust disease (Melampsoru spp.) is currently the most serious problem in willow biomass production in Europe. Other pests and diseases cause problems only on a local scale, though they could become more generally severe. Increasing clonal susceptibility to rust and an an increasing association of premature defoliation with lower rust severity levels have occutred over the period. A high degree of intrinsic variability in pathogen populations is expressed as fourfonnue specides (Espp.) and at least eight pathotypes of the most common rust species, M. epiteu. The same Espp. appear to occur among pathotypes in Sweden, New Zealand and the UK. A network of field experiments has been established, in collaboration with the Joint Trials Activity, to enable the rust pathotype composition to be compared between five countries. It also aims to identify an international set of standard willow clones to be utilized for characterizingrust pathotypes globally. KEYWORDS Diseases, Pests, Biomass crops, Energy crops, Willow, Poplar, Rust diseases, Melumpsoru, Septoriu, Chrysomelids, Biotechnology, Disease assessment INTRODUCTION The Pest and Disease Management Activity was established in 1989 in recognition of increasing problems that pests and diseases were beginning to cause within certain IEA Bioenergy Agreement countries in the production of woody biomass crops for energy. At this time there were many reports of investigations on pests and diseases of poplar, though not perhaps specifically in relation to energy production. Much was already known therefore about their identity and the problems they were likely to cause. In contrast, little was known about disorders of willows. Consequently, within the earlier IEABA Task II programme (1986-89),surveys had been initiated by the Willow Breeding Activity to identify the diseases and pests occurring in short-rotation willow coppice plantations and to make assessments of their importance (Hunter et al., 1988, 1989). Development of the surveys was considered necessary as a framework for the new Activity, in order that changes in the prevalence and severity of pests and diseases could be determined in relation to clone and site factors regionally, nationally and internationally. Initially, the surveys had identified the crucial importance of rust (Melampsoru spp.) as threatening the establishment and productivity of willow energy crops within the UK and Sweden, and work in the 45
46
D. J. ROYLE and M. HUBBES
Task V Activity began to analyze the rust population structure for variability with respect to different sites and the responses of different clones. Problems of willow rust are shared by Canada, Sweden, Eire, N. lreland, Scotland and England, and they have tended to dominate the work of the Pest and Disease Management Activity. This has led to studies on rust epidemiology, the scales of yield loss caused by the disease and in developing standard methods of rust assessment which would be suitable in both research and breeding programmes. There has been a close co-operation with the Joint Trials and Exchange of Genetic Material Activities with whom the Pest and Disease Activity has enjoyed two fruitful joint workshops, in Belgium 1990 and in Iowa 1991. Both of these Activities have themselves interests in pests and diseases, as factors in clonal performance in field trials and in relation to the development of resistance in breeding progmmmes. Within a separate section of the Activity, co-operative work on resistance mechanisms and biotechnology has been concerned with the development of tissue culture, isozyme and molecular and biochemical techniques for studies on Septoria musiva and bacterial canker of poplar and Melampsora rusts of poplar and willow. This report presents an outline of selected aspects of the Activity progmmme. INTERNATIONALSURVEYSOF WILLOWPESTS AND DISEASES, 1987.91
With the co-operation of observers in Canada, Eire, England, N. Ireland, Scotland and Sweden, annual surveys were done over 5 years on clonal willow plantations and plots (Table 1).
Table 1. Number of clone plantings assessed during surveys, 1987-91 -~__~~-__~~-_1_~~~~_~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~-~~~~~~~~~~~~~~~~ N. Ireland Scotland Eire Sweden Year England Canada 1987 13 93 5 94 25 1988 27 29 121 32 32 43 1989 30 39 141 36 80 30 1990 37 41 163 16 92 61 1991 27 37 176 16 _ _
Pests and diseases were assessed according to a simple questionnaire (Hunter et al., 1988, 1989, 1991) which was developed at Long Ashton Research Station where the surveys were co-ordinated. Recording was made simple to facilitate comparison of results from different observers, and severity classes assigned to different disorders were broad to emphasise extremes and to encourage standardizationin reporting. Symptom types were in 6 groups: (i) leaf rust, (ii) leaf and shoot tip dieback, (iii) disorders and cankers of woody stems, (iv) defoliation, (v) stool death, (vi) other/insect damage. Leaf infection due to rust was judged over an entire clonal planting as: 0 = no rust; 1 = low, occasional leaves infected; 2 = moderate, frequent leaves infected; 3 = severe, many leaves infected, rust pustules often in dense aggregations. Defoliation attributable to rust (potential yield loss) was scored as an estimate of the proportion of shoot lengths affected. Identification of disorders involving leaf and shoot-tip die-back could be difficult but was encouraged by listing various possible causes. As for leaf rust, stem disorders were gmded in 4 severity levels. Participants were asked to record possible causes of stool death, if known. Opportunity was given for individual comments on other, non-specific disorders, and on the general state of the plantation under scrutiny. As the surveys progressed, the relative incidence of feeding damage due to insects became apparent and, in the development of a computerized database, it was necessary to categorize beetle damage in a similar way to that for rust.
Diseasesand pests
47
Two assessments were done each year, in July/August and in September/October. Wherever possible, the same clone plantings were monitored over the 5 years. Whilst it was hoped that the questionnaire would be adopted by all patticipants, the observers in Canada and Sweden resorted to altemative ways of communicating their observations, mainly because of time pressums, though they were closely guided by the questionnaire.
At UK sites problems due to tbngal pathogens other than rust were rarely recorded, but in Canadian and Swedish plantings damage due to willow scab, Fusicludium saliciperdum, and branch cankers caused by Glomerella miyabeana, featured in older plantations. Damage by both these pathogens occur& mainly as shoot tip die-back and cankering of rods. In Canada, a common disorder on Sulix capreu, S. fragilis and S. alba hybrids was Marssoninasalicicola, attacking shoots and branches and causing leaf spotting. However, Fusicludium was the most serious problem, especially on S. lucida clones. At one site, Maple, Ontario, in 1990, 10/l 1 willow types were infected by this pathogen, only S. exigua remaining healthy. Many S. matsudunax S. alba crosses were also severely damaged, often with crown die-back. Swedish plantations also suffered from Marssonina leaf spotting, on S. caprea and S. fragilis type crosses. A die-back problem, otten associated with S. viminalisclones 78021, 78112 and 78183 and with S. daphnoides 79149, was identified in 1991 as associated with ice nucleation activity of the bacterium Pseudomonassyringue. This has become of increasing importance and is now serious with die-back progressing into rootstocks and causing stool death.
The most widespread damage from insects over the survey period was caused by Chrysomelid leaf beetles, e.g. Phyllodectavulgatissima. In the UK an average of 42% of plantings were affected, 8% severely so. Other locally damaging pests on mature stands included the willow weevil, Cryporrhynchus Zupathi,especially in Canada during the cool, wet summer of 1990. In 1991, damage by the potato leaf hopper, Empoasca fabue, was reported for the first time in Canada. In Sweden damage due to Terminalis midge on S. viminafisclones was consistently recorded and it also occurred to some degree in most UK plantations. The plant sucker, Aphrophoru sulicis, occurred in 1987 and 1988 on many S. butjuticu clones in the UK though it has not since been damaging. Eryophyid mite damage has also occurred on the same clones in UK plantings and on a number of clones in Sweden in 1991. Stem aphids, Melunoxantherumsalicis and Pterochforussp., were seen on many UK plantings in 1989and 1990, but not since.
Even though its intensity has varied between years and clones, rust has consistently been the most widespread, severe disorder in coppice willow plantations, especially in the UK and Sweden. Defoliation caused by rust has occurred frequently at many sites and on many clones and has almost certainly been responsible for heavy losses in biomass yields and progressive decline in the vigour of many plantings. UK In England rust incidence and severity was greatest in 1988 when more than 92% of clone plantings were infected, and over 60% had severe early defoliation. Most of this occurmd on S. bwjuticu clones, especially Korso which because of its extreme susceptibility is no longer regarded as of potential commercial value. Rust and associated defoliation has remained severe in many clonal plantations in recent years.
48
D. J. ROYLE and M. HUBBES
Even though there has been a gradual decline in the proportion of N. Ireland plantings afTected by severe rust and an increase in that affected by light attacks since 1988, defoliation has nevertheless increased and has thus appeared to be occurring at progressively lower rust levels. Over the entire survey period only 16% of clone plantings remained without rust, most of them (67%) the clone SQ83 (S. viminalis x triandra), which has so far remained resistant. In Scotland the frequency of rust early in the season increased after 1989, though at low intensities. Final seasonal severity and defoliation increased over the same period. This is reflected by an increase in rust on S viminalis clones in Scotland and elsewhere in the UK over the years. The data from UK plantings overall early in the season indicated trends towards an increasing proportion with moderate-severe rust over the 5 years. This is consistent with the disease appearing earlier or else developing at a progressively faster rate within plantings. These trends were not evident at the end of the season, however, the proportion without rust (13-20%) remaining fairly constant after 1988. Around 50% of plantings showed early defoliation in each of the last 4 years. In general, defoliation of S. viminalis clones occurred at lower rust severities than S. butjatica types. No cases of rust-induced defoliation were reported on 5. viminalis clones in 1987 but since then early rust attacks have led to defoliation. More early defoliation occurred on both S. viminahs and S. burjatica clones in 1988 than in other years, with 56% and 76% of plantings affected, respectively. Most records of rust in the surveys were of leaf infections. However, stem cankering caused by a new form of the rust pathogen has been increasing. The first records in 1988 were in only 8 plantings but, by 1991, 27/240 contained rust cankers. Overall, the S. viminalis clones, especially Bowles Hybrid (2 1.6% of plantings with cankers) were most severely affected. In 1991, several other clones were also affected, including the S viminalis clones Campbell, English Rod, Mullatin and Gigantea, and the hybrids S. x sericans, S. x hirtei, S. x dasyclados, S. x rubra and S. x calodendron.
Sweden. With the largest area of any country planted to biomass willows, Sweden has not repotted serious rust epidemics in recent years. In 1987 rust was most common on S. burjatica clones; exceptionally there were few problems on S viminalis types. As in the UK, many more leaf rust pustules had to occur on S. viminalis than on S. burjatica clones before senescence and leaf fall occurred. Less rust was recorded generally in 1988 and 1989 with infections occurring only late in the season. Even S. burjatica clones 056 and 075, which were infected severely before, were free of early rust at most sites. In 1990 there was again little rust except on S. viminalis 683 and some 15 S. caprea clones at Uppsala, and in some plantings in the south where other S. viminalis clones were severely attacked. There were few problems again in 199 1.
Canada. There has been a steady increase in the number of willow types with serious rust infections in Canada. In 1987 severe rust with defoliation did not occur until October and then at only one site on S. amygdaloides and S. pellita. This was also the case the following year with S. amygdaloides clones the worst affected and S. exigua x petiolaris and x eriocephala showing symptoms by September. Severe rust was detected earlier in 1989 with some S. amygdaloides clones defoliating by August at Maple, Ontario although many S. pellita and S. lucida clones were without tust. At two other sites only S. Iucida and S. discolor clones showed no rust throughout the season when it occurred on S. viminalis xpurpurea but not on the x triandra nor x caprea hybrids. Here several S. purpurea clones which were moderately infected in 1988 remained without rust in 1989. The same three sites were assessed in 1990 and 1991 but only at Maple late in the season was rust severe on two clones of S. amydaloides and two clones of S. exigua in 1990, and moderate on S. eriocephala and S. petiolaris in 1991. In general, rust in Canada is not as severe as in other countries and only relatively few clone types seem to be susceptible. Clone types originating from Europe and which were recorded as severe in the UK and Sweden, failed to exhibit high levels of rust and only 3/25 had any rust at all.
49
Diseases andpests
RUST VARIABILITYAND CLONAL RESPONSE Results from the surveys have indicated that perhaps the most difficult obstacle to the successful control of willow rust is the extreme variability of the pathogen The evidence suggests that variability is expressed at various levels: in the response of different clones, within and between different plantings of the same clone, and between different regions and countries. Within the Activity we set out to assess this variability in the UK, Canada and Sweden and to develop a standard set of differential clones on which forms of rust can be characterised.
Pathogenicity studies at Long Ashton, mainly using an in vitro inoculated leaf disc method, have shown that in the UK there are at least three species of Mehzmpsoru occurring on biomass willows: M. epitea var. epitea, the most widespread and common, M. caprearum and M. rib&i-viminalis. Furthermore, within the complex species M. epiteu var. epifea, five “form species (Espp.)” can be recognised within which there are at least eight “pathotypes”or “races”. These can be distinguished only by their reactions on differential willow clones (Table 2). The Esp. larici-epitea typica is represented by the 3 pathotypes LET1 (mainly infecting S. viminalis clones), LET2 (from the hybrid gcupreu x cinerea x viminalis 445 de Biardii) and LET3 from S. x culodendron. The Esp. larici-retusue is represented by the two pathotypes LRl, which causes the outbreaks on S. burjatica Korso, and LR2 from S. himalayas. The Esp. larici-daphnoides is represented by LDl from S. duphnoides Rubenima, and the Esp. ribesii-pwpureu by RP1 from S. purpurea Uralensis. Finally, the new “shoot-infecting”Esp. of rust from S. viminulis Bowles Hybrid occurs as a single pathotype, SIF. Table 2.
Proposed differential willow hosts and their reactions to pathotypes of Melampsora epitea var. epitea
Differential host viminalis ‘Mullatin x boydii x stipularis x calodendron butjatica ‘Korso’ himalayas daphnoides ‘Meikle’ purpureu ‘Jagellonica’
Pathotype LET1 LET2 + _ +
LET3
SIF -
LRl
LR2
LDI -
RPl
+ +
+ _ +
+ -
+
+
+
+
+
+
-
-
-
-
+
-
-
_
-
-
_
-
-
-
_
+
-
-
+
-
-
_
+ -
+
Since Suk species are either naturally distributed or introduced worldwide, it is perhaps not surprising that h4. epitea has been reported in Eurasia, N. America and recently Australia Exchange of information within the Pest and Disease Activity has suggested that in Sweden (Rams@, 1989) the rust pathogen shows five distinct patterns of pathogenic@ when tested with S. &phnoides “ESO”,S. viminalis x caprea, and several clones of both S. viminalis and S. x dapyckrdo (The name S. x dusycludos is used on the continent to refer to two forms called in the UK S. bmjutica Nasarov (synonym S aquatica grundis, see Bean (1980)) and S. x &sycluui~ Wimm. (Stott, 1991)). The Swedish pathotypes seem to belong to the form species larici-epitea typica, laricidaphnoiaks and possibly Zarici-retusae. In New Zealand, rust infection was observed for the Erst time on S. incuna x duphnoides in 1978. This was a new rust apparently similar to M. epiteu which caused severe attacks on S. viminalis and hybrids in 1985 (Spiers and Hathaway, 1988). From the host lists in New Zealand, the iU. epitea rusts appear to belong to larici-daphnoiaks and larcici-epitea @pica.
50
D. J. ROYLE and M. HUBBES
Since it could be dangerous to exchange isolates of the airborne rust pathogen between countries for pathogenicity testing, a network of five field trials was established in March 1991, in collaboration with the Joint Trials Activity, at Long Ashton, Loughgall, N. Ireland and Craibstone, Aberdeen, Uppsala, Sweden and Ontario, Canada. Each trial incorporates small (5 x 5 stool) plots of 24 clones, (12 from UK, 6 from Canada and 6 from Sweden), replicated five times and selected according to a range of responses to rust in each country. The project aims to compare rust populations in order to determine which pathotypes occur in the different countries. It also aims to establish the suitability of the clones for studying pathotype x environment interactions. From this we hope to develop an international set of standard willow clones for characterizing rust pathotypes globally. In the establishment year, data has already indicated a different spectrum of rust pathotypes between the countries. DISEASE ASSESSMENT Field research on biomass diseases, the IEA rust/clone trials in particular, demands standardized methods for monitoring disease levels within plantings. Also, there will be an increasing need for monitoring as interest in growing amble energy crops increases. The underlying reason for this monitoring will govern the scale, frequency and required accuracy of the assessments that are made. However, assessments that are unrepresentative of the planting are worthless, whatever the reason for the assessment. The reliability of any attempt to quantify disease is strongly influenced by the protocol used to obtain the samples, and the accuracy and precision (defined by Campbell and Madden, 1991) of the visual disease severity assessments. These factors have been investigated in a 6-month progmmme at Long Ashton, within the IEA Activity, and utilizing the co-operation of participants in the rust/clone trials. The study focused on willow rust because of the priority need to develop standarized methods of assessing this disease.
The spatial and temporal spread of rust was investigated in a small plot (12 x 19 stools) of S. dasyc1udos Wimm. A single shoot was randomly selected for every stool, and rust incidence and pustule frequency on every 20th leaf were recorded weekly. Preliminary analysis using mapping suggested that the disease spread was not focal. However, further analyses will be required to confirm this and more work is needed if recommendations about sampling are to be provided with confidence.
It is widely accepted that disease assessment keys can improve the reliability of visual estimates of disease severity, so it perhaps follows that they are essential to good assessment. A simple key for willow rust assessment was produced by McCrackenand Dawson (1992). However, currently, no key has gained wide acceptance and so the first aim of this study was to discover whether any key was necessary. Method. A large number of leaves of S. burjuzicuKorso were collected from the IEA rust/clone trial at Long Ashton. Of these, 30 were selected which provided a range of rust severity typical of that found in the field. Professional photographs were produced of the abaxial surtbces of the leaves, labelled and placed singly in transparent cellophane sleeves. A set of photographs, with score sheets and instructions, was mailed to each of the other four groups participating in the rust/clone trials. Observers were instructed to use a hand lens (x10) when making an assessment. There were several
Diseases and pests
51
observers from each group, including Long Ashton, who participated. Objective estimates of disease severity were obtained using video image analysis. Objective and visual estimates of disease severity were logit transformed to stabilize variance. Least squares regression models were obtained for each observer for visual fitted against objective estimates. The similarity of these models was compared in the way described by Parker (1991). Results. Even observers from the same research group were found to differ greatly in their perceptions of disease severity (p>O.UZ). For most observers the errors in assessment were attributable to gross over-estimation of severity. This problem has been reported for visual assessments of brown tust of barley (Puccinia hordei) by Beresford and Royle (1991). Over-estimationwas a particular feature of the assessments of observers who tended to ascribe severity into bands rather than on a linear scale. It is suspected that such individuals generally made assessments with reference to disease scales. The evidence obtained indicated that assessment into bands should be discouraged because it was apparent that even gross differences in disease intensity could be missed. A key previously untested (Parker, 1991) was refined to correspond to the shape and size of willow leaves and tested for its efficacy in improving the reliability of assessments. The key is based on a generic leaf grid made-up of 1% sectors, The coordinates of this grid were incorporated into a computer programme that includes a scaling feature so that leaf dimensions (length & breadth) can be inputted allowing any size of leaf to be generated. Three such leaf grids printed on acetate and covering the range in leaf size of the photographs were provided to the same groups for a second assessment. Observers were instructed to use this key as an overlay, Estimates provided by different observers were, in general, more similar in the second assessment than previously achieved (Fig. 1). Moreover, vast over-estimation was eliminated, so the assessments were more accurate. The effect on assessment precision was less clear. For several observers there was an obvious improvement, but for others improvement was either marginal or, in a few cases, precision was slightly reduced. Further work is necessary to clarify the effect of using the key on assessment precision. Several observers indicated that they found the use of the overlayed key either difficult or too time consuming. We propose that the key should be printed onto paper and used as a guide at the start, and subsequently as necessary, for each assessment. It would be prudent to undertake further investigation to ensure that the key is effective when used in this way. RESISTANCEMECHANISMSAND BIOTECHNOLOGY This part of the Activity programme aimed to develop tissue culture and in vitro techniques for Sepzotiu m&vu and bacterial canker on poplar and Melampsoru spp. on poplar and willow. It also compared pathogen isolates using isozyme and other biochemical methods and investigated resistance mechanisms by molecular and biochemical techniques. Septoriu musivu infects native and hybrid poplars throughout N. America. Originally it was considered to be endemic in Populus bulsuminrjkra,stands but when hybrid poplars were used in plantings it developed into a lethal canker disease on many clones. In the mid-1970’shybrid poplar plantations in the north central USA were badly damaged by S. musivu (Ostry and McNabb, 1985). Hybrid poplars are expected to play an important role in future energy plantations and the impact of Seproriu canker must be determined and control methods developed (Hubbes and Wang, 1990). Cooperation between scientists in Belgium, Canada and the USA within progmmmes of this and the Exchange of Genetic Material Activities has resulted in significant progress being made towards this goal.
52
D. J. ROYL!?and M. HKJBBES
Since S. musivu does not occur in Europe, genetic poplar material is being exchanged to test its field response to the pathogen (Strobl er al., 1990, Mottet and Vallee, 1990). Simultaneous inoculations in Quebec and Ontario of 150 clones from Belgium, with two Seproriu strains, are also being done. Several clones with strong resistance to the disease have been found. Tissue culture and new molecular biotechnology techniques offer many advantages for poplar improvements over field screening tests (Ostry, 1991). The discovery that somatic variation can be induced and recovered by the passage of plant cells through a tissue culture cycle has opened up a new field of investigation. A sensitive in vitro bioassay was developed for assessing the resistance of poplar clones to Sepzoriu leaf spot (Ostry et al., 1988). The results correlated well with those from previous field trials. A recent study involved the occurrence and extent of phenotypic variation in Melumpsoru rust resistance in Pop&s deltoides somaclones generated from leaf callus. A race-specific interaction was found among regenerates for the latent period and uredia per unit leaf area (Ostry, 1991). The importance of the magnitude of variability in pathogenicity within a pathogen population has already been noted, in regard to willow rust. This is also important in developing resistance through somaclonal variation and in screening for resistance in conventional breeding programmes. S. m&vu isolates have been grouped by isolation of the pathogen’s mitochondrial, ribosomal and nuclear DNA (Hubbes and Wang, 1990). According to these authors, RFLP’s and DNA fingerprinting give best results, showing complete homology between several isolates. Strains that are homologous in their fingerprint are identical, regardless of the clonal host from which they are derived. No linkages have been found between the RFLP banding pattern and strainal virulence. Expansion of this work is planned, with larger samples, since isoxyme analysis has identified some isolates with a different banding pattern (Ostry, 1991). Another approach to develop disease resistant material is to engineer “subtle resistance genes”, such as proteinase inhibitors, into the target plant. McNabb et al. (1990) hypothesize that using a gene that codes for the production of a proteinase inhibitor would generate the stable resistance desired for insect and pathogen management strategies. Several poplar hybrids have been transformed with Agrobucterium fumefuciens strain A281 via four binary vector plasmids (McNabb et uZ., 1990). To date, 200 different putative transformed clones carrying components of the proteinase inhibitor II gene have been produced. One component, the wound inductive promoter, was used with the reporter CAT gene. Systemic promoter mRNA has shown the promise of using wound-inductive promoton as regulators for other resistance gene constructs. It is hoped that the proteinase inhibitor is also active against the proteinases of S. musivu. Bioassays showed encouraging results (McNabb ez al., 1990).
ACKNOWLEDGEMENTS The work of the Pest and Disease Management Activity has been possible only with the considerable contribution and support from a number of people. In particular, we are grateful to Mr.T. Hunter for co-ordinating the surveys, Dr. M.H. Pei for the pathogenic@ testing and Dr. S.R. Parker for supervising the disease assessment work We also thank all those who co-operated in collecting data for the surveys and in the disease assessment exercise, especially B. Beatson, M. BuIfIn, M. Dawson, J. Ford-Robertson and M. Ramstedt. Funding by the Energy Technical Support Unit for the Department of Energy for the UK rust/clone trials and for other aspects of the research at Long Ashton is gratefully acknowledged.
REFERENCES Bean, W.J. (1980). Trees and Shrubs Hardy in the British Isles. VoLIV. John Murray, London Beresford, R.M. and D.J. Royle (1991). The assessment of infectious disease for brown rust (Puccinia hordei) of barley. Plant Path., 4Q, 374-38 1.
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Campbell, C.L. and L.V. Madden (1990). Introduction to Plant Disease EpidemioZogv John Wiley and Sons Inc., USA. Hubbes, M. and CL. Wang (1991). Strainal identification of Septoria musiva. In: Proceedings of the Joint Meeting of the IEA Task 5 Activity Groups on Exchange of Genetic Material, Joint Triab of Aldus, pormlus and &li~ and Pest and Disease Management, 1990, Geraardsbergen, Belgium, ~~43-56. Hunter, T., D.J. Royle and K.G. Stott (1988). Diseases of Saliwbiomass plantations: results of an international survey in 1987. In: Proceedings of the IEA/BA Task 2 Workshop Biotechnology Development, 1987, Uppsala, Sweden, pp.37-48.
Hunter, T., D.J. Royle and K.G. Stott (1989). Diseases of Salti biomass plantations: results of an international survey in 1988. In: Proceedings of the ZEA/BA Task 2 Workshop WiZlow Breeding and Biotechnology Development, 1988, Long Ashton, Bristol, UK, pp.68-75.
Hunter, T. and D.J. Royle (1991). Diseases and pests of Saliwenergy plantations: international survey results in 1989 and 1990. In: Proceedings of the Joint Meeting of the IEA Task 5 Activity Groups on Exchange of Genetic Material, Joint Trials of k pomtlus and && and Pest and Disease Management, 1990, Geraardsbergen, Belgium, pp.27-3 1. McCracken, A. and M. Dawson (199 1). Clonal response in Salix to Melampsora rusts in short rotation coppice plantations. Eur. J. For.(in press).
McNabb Jr., H.S., N.B. Klopfenstein, S.A. Heuchlin, and R.W. Thornburg (1990). The integration of genetic engineering into Iowa State University’s woody biomass research program. In: Proceedings of the Joint Meeting of the IEA Task 5 Activity Groups on Exchange of Genetic Material, Joint Trials of A.&s,, poDulus and && and Pest and Disease Management, 1990, Geraardsbergen, Belgium, pp.66-77. Mottet, M.J. and G. Vallee (1990). Belgium poplar exchanged with Quebec, Canada. In: Proceedings of the Joint Meeting of the IEA Task 5 Activity Groups on Exchange of Genetic Material, Joint Trials of & Populus and &_& and Pest and Disease Management, 1990, Geraardsbergen, Belgium, pp.97-103. Ostry, M.E. (1991). Somatic variation in disease resistance of poplar. In: Proceedings of the Joint Meeting of the IEA Task 5 Activity Groups on Exchange of Genetic Material, Joint Trials of Alnus, Popuius and Salix and Pest and Disease Management, 1991, Iowa, USA. In press. Ostry, M.E., R.E. McRoberts, K.T. Ward and R. Resendez (1988). Screening hybrid poplars in vitro for resistance to leaf spot caused by Septoria musiva. Plant Dis., l&497499. Ostry, M.E. and H.S. McNabb Jr., (1985). Susceptibility of Populus species and hybrids to disease in the north central United States. Plant Dis., @, 755-757. Parker, S.R. (199 1). Studies on some factors influencing the reliability of disease measurements in winter wheat crops. Ph.D. thesis, University of Bristol, UK. Ramstedt, M. (1989). Identification and characterization of pathogens important in Swedish Salix plantations. In: Proceedings of the ZEA/BA Task 2 Workshop WillowBreeding and Biotechnology Development, 1988, Long Ashton, Bristol, UK, pp. 82-87. Spiers, A.G. and R.L. Hathaway (1987). Melampsora rusts of Salt&in New Zealand. In: Proceedings of the IEA/BA Task 2 Workshop Biotechnology Development, 1987, Uppsala, Sweden, pp.6569. Stott, K.G. (199 1). Nomenclature of the promising biomass coppice willows, Salix x sericans Tausch ex Kern., Salix dasyclados Wimm. and Salix “Aquatica Gigantea”. Bot, J Scotl., 46, 137-144.
Strobl, S., K. Fraser and R. Tavares (1990). Belgium poplar exchange with Ontario, Canada In: Proceedings of the Joint Meeting of the IEA Task 5 Activity Groups on Exchange of Genetic Material, Joint Trials of Alnus, Populus and Salix and Pest and Disease Management, 1990, Geraardsbergen, Belgium, pp. 104- 113.
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D. J. ROYLE and M. HIJBBES
54
6
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-8 -6
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Clogit)
Fig. 1. Comparison of the assessments made by three observers from one site collaborating in the IEA nut/clone interaction experiments (a) without the new key, and (b) with the new key as an aid.