breeding against dutch elm disease adapted to the mediterranean climate
TRANSCRIPT
Euphytica (2008) 163:45–56
DOI 10.1007/s10681-007-9573-5Breeding against Dutch elm disease adapted to the Mediterranean climate
Alberto Santini · Nicola La Porta · Luisa Ghelardini · Lorenzo Mittempergher
Received: 29 June 2007 / Accepted: 10 September 2007 / Published online: 20 September 2007© Springer Science+Business Media B.V. 2007
Abstract Breeding for resistance was consideredone of the possible ways to limit the damages causedby the epidemics of Dutch elm disease (DED) duringthe last century. The elm breeding program developedby CNR in Italy was based on the idea that the Medi-terranean environment would need its own speciWcselections. A base broadening of the genetic resourceswas operated. A base of native elms with a set of goodcharacters to act as parents was bred with diVerentAsian elm species that showed the ability of acclima-tization to the diVerent climates in which elms have tobe planted. For this aim a large collection of elmspecies was constituted, followed by hybridisationstudies. Progenies were tested for DED resistance.The more resistant clones were planted in trial Weldscharacterised by contrasting Mediterranean climatesin order to select the best potential genotypes adaptedeither to coastal or to mountain environmental
conditions. More than 60 clones resistant, fast-grow-ing and showing remarkable aesthetic ornamentalcharacters were obtained.
Keywords Dutch elm disease · Breeding · Resistance · Elm · Mediterranean climate
Introduction
Starting from around 1910 in Northwest Europe twodestructive pandemics of Dutch Elm Disease (DED)caused by the introduction of two very diVerent fun-gal pathogens, Ophiostoma ulmi (Buisman) and O.novo-ulmi (Brasier) aZicted the elms in Europe and,20 years later, in North America (Brasier 2000;Guries 2001). The gravity and impressiveness of thedamages caused by the disease stirred up the interestof public opinion and researchers to explore solutionsto the problem (Rohring 1996).
The idea of searching for resistance to DED in theEuropean elm species and elm populations, associ-ated with the hope of enhancing resistance throughbreeding, arose in 1928 at the Willie CommelinScholten Phytopathological Laboratorium in Baarn(The Netherlands), (Heybroek 1993, 2000), where awide collection of elms was constituted and the Wrstresistant cultivars were obtained within the Euro-pean species.
Following the Dutch example, several elm breed-ing programmes in Europe were set up in several
A. Santini (&) · L. Ghelardini · L. MittempergherIstituto per la Protezione delle Piante – C.N.R, Via Madonna del Piano, 10 50019 Sesto Worentino, Italye-mail: [email protected]
L. Ghelardinie-mail: [email protected]
L. Mittemperghere-mail: [email protected]
N. La PortaIASMA Research Center Department of Natural Resources, Via E. Mach 2, 38010 S. Michele a/A, TN Italye-mail: [email protected]
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countries beginning from the late 1970s (Mittemper-gher and Santini 2004) when the second and moredestructive pandemic of DED caused by O. novo-ulmiwas invading the Continent. Elm clones developed inHolland in the ‘30s did not show suYcient resistanceto this new pandemic and no complete resistance wasever found in European and American native elm spe-cies, although in second or third generation hybridclones of purely European elms suYcient resistancecan be accumulated (Heybroek pers. comm.) But theuse of Asian species with their higher original resis-tance would speed up the process. Further, several2nd generation Dutch and also American resistantclones, simple or complex hybrid between native andAsian species, were developed and delivered on themarket (Rohring 1996; Guries 2001). An elm breed-ing program adapted to the Italian conditions startedin 1975 and is still going on in Florence by the Insti-tute of Plant Protection of C.N.R. The idea underlyingthis project was the conviction that the Mediterraneanenvironment would need its own selections (Mittem-pergher and Santini 2004). The favourable adaptationof Asian species in Italy, like Siberian elm (U. pumilaL.) and others, and the unsuitability of the Dutchselections to the hot and dry areas of central Italyencouraged to test the Asian species both in order toassess more in depth their adaptability to the Mediter-ranean climate and to broaden the genetic base of thenative species.
Selection of superior genotypes reduces geneticvariation in cultivated species (Simmonds 1993;Tanksley and McCouch 1997). However, whenbreeding is designed for obtaining plants adapted todiVerent environmental conditions and for diVerentuses, the outcome could result in an increase of vari-ability (Cox and Wood 1999). The case of elm breed-ing for resistance to DED is paradigmatic. The uses ofelm are manifold: from wood to fodder, from medi-cine to urban silviculture and many others. For thisreason breeding for resistance is not enough: manyother features are requested as tree silhouette, fastgrowth, leaf and bark colour, leaf shape and dimen-sions. In order to satisfy all these needs, maintainingat the same time enough genetic variability to buVerthe rising of possible new stresses, such as new formsof the disease or other diseases and climate change,we decided to operate a base broadening of thegenetic resources, or, as it will be called later, “incor-poration” (Simmonds 1993). A base of native elms
with enough good characters to act as parents wasbred with diVerent Asian elm species that showedability of acclimatization to the diVerent climates inwhich elms have to be planted as well as a fair levelof disease resistance.
One of the problems was the crossability amongspecies (Mittempergher and La Porta 1991) followedby a reliable and repeatable method of inoculation,and the availability of experimental Welds located indiVerent and contrasting environmental conditionswhere the elms could grow for years.
In this paper we present the results of the 30-yearprogramme on elm breeding built up with the aim ofobtaining DED resistant elm varieties that accom-plished the remarkable aesthetic ornamental charac-ters with adaptability to the Mediterranean climateconditions. Because of their fast growth, adaptabil-ity to highly diVerent soil conditions, their resis-tance to wind, to pruning and root damage, to cityconditions in general, elm trees are ideal trees forcity and landscape. So there is a big renewed inter-est in elms in the tree planting community that isalso highly demanding in terms of new resistantclones. Moreover, since other species that were typ-ically used for urban plantations, such as plane tree(Platanus hybrida Brot.) and Horsechestnut (Aescu-lus hippocastanum L.), are now subjected to diVer-ent diseases, so planters want to reduce risks. Inprevious papers we presented four resistant hybridelm clones already patented and released on themarket: ‘S. Zanobi’, ‘Plinio’ (Santini et al. 2002),‘Arno’ and ‘Fiorente’ (Santini et al. 2007). Here wereport the results of the crosses between pure speciesand of adaptation trials to contrasting Mediterraneanclimates.
Material and methods
Collection of material
In late 70s a wide collection of elm species and prove-nances from all over the world with a particular pref-erence for Asian species, generally more resistant toDED (Smalley and Guries 1993) was set up. Collec-tions were established with two aims:
(1) To check adaptation to a new environment, con-stituted by the Mediterranean climate and by allthe biotic and abiotic damage agents that may
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aVect introduced species under these conditions,and, as a consequence the new hybrid clones;
(2) to obtain adult plants for breeding work.
Hybridisation studies
Crossability among elm species which involved theuse of several European and Asian species of diVerenttaxonomic sections (see Table 1) was checked underMediterranean climatic conditions. Ten pure speciesplus an U. £ hollandica ‘Belgica’ were crossed witheach other for a complete full diallelic cross of 121crosses. The mother trees, as well as the trees used aspollen donors, were grown in three diVerent orchardslocated near Florence and Pisa.
Pollen of the desired species was obtained from cutbranchlets held in vases with water during the pollendispersal phase. DiVerent species and individualswere kept in separate rooms of a greenhouse to avoidcontamination. The collected pollens were conservedat 3–4°C and dehydrated at 10% RH, when usedwithin a short period of time (few days or weeks).When pollen had to be stored for 6 months (AsianAutumn Xowering species) or for about 1 year (tocross the later pollen donor with the earlier springXowering species), it was conserved at ¡20°C and10% RH. Pollen vitality was checked before pollina-tion by using the Fluoro-chromatic Reaction tech-nique (Heslop-Harrison and Heslop-Harrison 1970;Heslop-Harrison et al. 1984).
Flower pollination was carried out by injectingpollen into the pollination bags with forced air. Atleast three bags were used for each single cross, aswell as, at least three control bags on each mother treedid not receive foreign pollen in order to check theselWng for each mother tree. Matured seeds were har-vested and sown in open air nursery beds, monitoringgermination. The obtained seedlings were checkedmorphologically during the Wrst and second growingseason in order to ascertain their hybrid nature(Table 2). The percent of viable seedlings on theamount of full seeds was scored at the end of the Wrstgrowing season. Percentages were arcsin transformedand subjected to separate ANOVA for each motherspecies in order to evaluate the cross eYciency. Sig-niWcant diVerences were assessed by LSD test.
Screening disease resistance
The obtained seedlings were evaluated for DED resis-tance. Selection for resistance was operated followinga two step protocol:
(1) A 3-year-old elm seedlings obtained by con-trolled crosses were grown in the I.P.P. nurseryand planted in the Weld. The following year, dur-ing the third week of May, which is the time ofbeetle Xight in the area of Florence, trees wereinoculated. Inoculation was performed with a sin-gle wound per plant, using a knife blade carrying
Table 1 Elm species utilized in the frame of the C.N.R. breeding program for elm resistance to Dutch elm disease. The unique Med-iterranean species has been underlined
Where ¡ = not resistant; + = resistant; ++ highly resistant
Section Species Common name Nb. parent trees used Origin of the parent trees DED Resistance
Blepharocarpus U. laevis Pall. European white elm 2 France ¡Ulmus U. minor Mill. European Weld elm 5 Italy ¡
U. glabra Huds. Wych elm 4 Italy ¡U. pumila L. Siberian elm 13 Turkestan, W Siberia ++
U. japonica Sarg. Japan elm 6 Japan +
U. wilsoniana Schn. Wilson elm 1 China ++
U. elliptica Koch. Armenian elm 1 Caucasus ¡U. £ hollandica Mill. Dutch elm 1 The Netherlands ¡cv. ‘Belgica’
Microptelea U. parvifolia Jacq. Lacebark elm 5 Korea, Japan ++
n.d. U. chenmoui Cheng Chenmoui Elm 5 NE China +
U. villosa Brandis Cherrybark elm 3 Himachal Pradesh +
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two drops of a 0.2 ml of a 1 £ 106 ml¡1 yeastphase cells, consisting of two tester isolates of thesubsp. novo-ulmi and subsp. americana of O.novo-ulmi (Brasier and Kirk 2001), so that theinoculum would be absorbed by the tree’s risingsap. By putting the suspension drops on the knifeblade before making the incision the suspensionis suckled in by the under pressure in the vesselsand there is no clogging by air.
(2) Disease assessing. Symptoms of disease (per-cent of defoliation and percent of dieback) wereobserved after 4 weeks and at 3 and 8 monthsfrom inoculation date by three independentassessors. Seedlings presenting less than 10%dieback were vegetatively propagated by hard-wood cuttings and planted out the followingyear according to a randomized block design.Clones showing less than 25% of dieback wereconsidered resistant and evaluated for othercharacters.
(3) Twelve rooted cuttings per clone, divided intothree blocks, were used. Inoculations and diseaseevaluations were performed 2 years after, asdescribed above and the symptoms, were com-pared with those expressed by clones havingknown DED responses acting as boundary mark-ers, generally the Dutch clones ‘Commelin’ and
‘Lobel’ highly susceptible and intermediatelyresistant, respectively.
Adaptation trials
The following traits were also evaluated in diVerentenvironmental condition in order to check the possi-ble eVect of phenotypic plasticity and which are thebetter condition for each clone growth. In order tocheck these traits two adaptation trial Weld wereplanted in 2000 in diVerent climates in Italy. In eachWeld a randomized block design was followed withpossibly three blocks of four ramets for each clone.Traits were measured each year and a Wnal evaluationwas performed in 2006.
Clones showing DED resistance were also evalu-ated for:
(1) leaf shape: length, breadth and slenderness, thefavourite shape being that of U. minor leaveswhich are rounder than the Asian elms.
(2) leaf colour: dark green was considered preferablefor being similar to the native Weld elm
(3) shape of the crown: columnar with a monocormicstraight trunk and slender branches is the favour-ite shape.
(4) height and diametric growth.
Table 2 Morphological features taken into account in order to check the hybrid nature of the elm seedlings obtained after the hybrid-ization experiments
Species Morphological characters investigated
U. laevis Pall. Leaf main teeth uncinate, leaf base strongly uneven, winter buds slender, acute, presence of a white coat below the bark.
U. minor Mill. Leaves oblanceolate to nearly circular, smooth and glossy above, glabrous beneath with axillary pubescence, base uneven, corky wings,
U. glabra Huds. Leaves large, ovate or elliptic to obovate, frequently 3 lobed at the apex, young shoots red-brown and thickly pubescent, winter buds big and dark brown
U. pumila L. Leaves elliptic to lanceolate, primary teeth with a few small secondary teeth, base usually even, young shoots thin, grey-brown, winter buds small
U. japonica Sarg. Leaves elliptic to obovate, lighter beneath and soft pubescent, reddish in winter, shoots occasionally with corky ridges.
U. wilsoniana Schn. Leaves elliptic to more obovate or ovate; young shoots soft pubescent
U. elliptica Koch. Leaves large, elliptic-oblong, smooth above, slightly pubescens beneath; young shoots pubescent.
U. £ hollandica Mill. Leaves medium to large, oval-elliptic, smooth above, base very uneven
U. parvifolia Jacq. Leaves small elliptic, leathery and tough; bark exfoliating in large rounded pieces
U. chenmoui Cheng Leaves large obovate to oblong, velvety, scabrous, abaxially densely pubescent, adaxially densely hirsute and pubescent along midvein, base uneven.
U. villosa Brandis Young shoots yellowish red, leaves obovate to oblong, only slightly asymmetrical, lenticels on shoots.
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Clones were ranked for these traits and resistanceaccording to a 5 step scale by three independentobservers. This datum is reported together with theclone code as a variable number of marks. The scalegoes from no marks = not eligible clone, to fourmarks = clone that accomplish all the requested char-acters: resistance, adaptation, leaf colour, trunk andcrown shape.
Site A is located in the experimental farm “LaTorre di Feudozzo” (AQ) managed by the UYcioTerritoriale per la Biodiversità—Castel di Sangro ofthe Corpo Forestale dello Stato located at N 41° 45’ E14° 25’; 960 m asl on the Apennines and is character-ised by a mountain Mediterranean climate with coldand snowy winters and cool summers without asevere drought period. Average rainfall is 1520 mm/year with a peak in Winter. The yearly average tem-perature is 9.6°C. The coldest month is January withan average minimum temperature ¡0.9°C.
Site B is located in the experimental farm “LaMarsiliana” (GR) UYcio Territoriale per la Biodiver-sità—Follonica of the Corpo Forestale dello Statolocated at N 43° 01’; E 010°48’; 300 m asl and ischaracterised by a typical Mediterranean climate withmild and moist winters and by hot and dry summers.Average rainfall is 760 mm/year, with a peak inAutumn. The yearly average temperature is 15.3°CThe hottest month is August with an average maxi-mum temperature of 29°C.
Results and discussion
Hybridisation studies
In Mittempergher and La Porta (1991) we reportedthe percentage of viable seeds obtained by thecrosses, here we present the percent of viable seed-lings for each cross (Fig. 1) on the amount of fullseeds.
It was not possible to carry out a full diallelic crossamong the 11 species used. Out of the 121 forecastedcrosses (110 plus 11 controls), only 85 (75 plus 10controls) were positively performed, representing a70% of success. This result was mainly due to thelocation of the mother trees, the availability of viablepollen at the moment of the female receptivity, theopen air unfavourable climatic conditions during andafter Xowering, as heavy snowing and late spring frost
that can cause the embryos abortion. This is the casewith U. villosa, an Himalayan species, which allthrough the three years of crossing trials did not pro-duce seed in any cross. In the Mediterranean climatethe mild January or early February conditions favourits Xowering but later, it suVers for recurrent latefrosts. Therefore, this species was used only as pollendonor (Fig. 1). Because of its uncertain taxonomicposition, an incompatibility phenomenon cannot beexcluded.
The number of seedlings per positive crossing wasvery variable from a few, up to over 1,500, with ahigher frequency of around 100–400.
Unilateral crossing barriers were observed formany species combinations so that success of severalcombinations depended on male–female interactionand the parental individual. Hybrid seed productionwas generally related to taxonomic aYnity of theparental species.
The control for selWng percentage was in generalzero or close to zero. Self-incompatibility is a com-mon phenomenon in Ulmus, which makes breedingpossible without requiring emasculation. Some spe-cies are exceptions, particularly U. japonica andU. parvifolia, where selWng percentage of the totalpollinated Xowers reached the average of 3.8% and4.5%., respectively. As for the latter species, whichXowers in the late summer, autumn, a remarkable var-iability in selWng was found among the individualtrees ranging from 0.2% to 7.8%. In species in whichselWng percentage is so high, as in U. parvifolia, theavailability of a hybridisation molecular markerwould play a crucial role.
When the European species are concerned the mostsuccessful results were obtained when U. glabra andU. minor were crossed as pollen donors withU. pumila and U. japonica. Encouraging results werealso obtained by crossing U. glabra as mother treewith U. japonica. On the other hand, U. laevis wasthe most recalcitrant species especially when used asmother tree. This result is explained by the taxonomicposition of this species, that naturally grows in North-East Europe but belongs—together with the Ameri-can elm (Ulmus americana L.) to the separate taxo-nomic section—Blepharocarpus The only exceptionwas when U. laevis was crossed with U. villosa,whose taxonomic position remains so far uncertain(Table 1). This cross was very successful—6.9% ofthe pollinated Xowers produced 1653 seedlings vs.
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Fig. 1 Results of hybridization experiments. Data refer to per-centage of seedlings on full seeds; in black are mean values ofselWng. An asterisk (*) denotes presence of characters of the
male parent on some of the seedlings of the hybrid progeny.Means not sharing the same letter diVer according to LSD test
U.parvifolia U.minor U.glabra
3.3 b
2.3 b
1.9 b
0.6 a
3.6 b
6.8 c
7.8 c
8.8 cd
11.1 d
3.8 b
0 4 8 12
Se
*JA
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*CA
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19.6 d
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0.9 a
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0.8 a
0 10 20 30
Se
*JA
*CH
WI
EL
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*CA
PU
LA
U.japonica U.laevis U.pumila
4.6 a
32.9 e
18.1 d
14.5 cd
14.0 cd
12.6 bc
10.8 b
10.4 b
10.1 b
0 10 20 30 40
Se
CA
HO
LA
*PA
*GL
*CH
*PU
*VI 0.1 a
0.2 a
0.3 a
0.4 a
0.6 a
0.6 a
0.6 a
0.9 a
6.9 b
0.5 a
0 2 4 6 8
Se
*VI
PA
WI
GL
PU
CH
CA
EL
JA
42.9 d
0 a
0.3 a
2.7 b
5.6 b
12.5 c
0 15 30
Se
*GL
*CA
*VI
*WI
*PA
LA
0.1 a
45
U.wilsoniana U.chenmoui U.elliptica
0.1 a
0.2 a
0.5 a
1.0 a
3.3 b
5.1 b
0 1 2 3 4 5 6
Se
EL
GL
*PU
*VI
*PA
0.1 a
0.3 a
0.4 a
8.0 b
0 3 6 9
Se
*WI
GL
VI
PU
PA
14.1 d
11.5 c
11.1 c
11.0 c
9.7 c
3.1 b
0.8 a
0.2 a
0 6 12
Se
*CH
HO
WI
GL
*JA
PA
*VI
U.X hollandica U.villosa
0.2 a
0.1 a
0.3 a
0.3 a
8.6 b
16.0 b
0 7 14
Se
*JA
*CH
LA
EL
PA
VI
PU
GL 0 a
0.3 a
0.8 a
2.8 b
3.6 bc
5.6 c
6.9 c
10.1 d
11.3 d
0.5 a
0 4 8
CA
JA
LA
PU
PA
GL
EL
WI
CH
HO
12
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Euphytica (2008) 163:45–56 51
0.5% of the control and 0.9 of the best of the otherpollen donor species. Despite the seeds presented alower development of the endosperm compared withthe open pollinated ones, as it is the case of someother crosses (Lopez-Almansa 2004) seedlings evenshowed hybrid vigour. However, after about threemonths of growth, when the seedlings were 10–15 cmhigh and beared about 6 pair of fully expanded andnormal leaves, they suddenly began to wilt and dry upand none survived the following year. We attemptedthis cross for three successive years using three diVer-ent female parents and obtained the same result. Alsogrowing seeds from this cross in vitro did not alter theresult. A similar case was also reported by Sholtz(1957) who crossed U. thomasii with U. pumila.Hybrid lethality was also observed in other species asNicotiana interspeciWc hybrids (Kobori and Marub-ashi 2004; Tezuka and Marubashi 2006), in Ipomea(Diaz et al 1996) and Gossypium (Phillips 1977).This delayed incompatibility did not occur whenU. villosa was used as pollen donor with other species(Fig. 1).
Screening disease resistance
The results of this activity are summarised in Fig. 2where the total number of clones found resistant at theWrst (353) and second step (123), is reported and theincluded clones are grouped according to their paren-tal species. Only one third of the clones that resulted
resistant at the Wrst step of the selection process, wereconWrmed to be resistant at the second step, accordingto the adopted protocol. Among those resistant clonesabout 60 resulted both resistant and showing someother valuable features (Fig. 3, Table 3). The crossesbetween U. japonica. used as female with U. pumilaand U. £ hollandica. with U. pumila gave the highernumber of resistant clones, 15 and 14, respectively.U. wilsoniana. £ U. pumila, and U. pumila £ U. minorcrosses gave Wve and four clones respectively. One ofthese last four has recently been patented andreleased on the market with the name ‘Fiorente’(Santini et al. 2007). Three clones were obtainedfrom each of the crosses U. £ hollandica £U. wilsoniana, U. parvifolia £ U. pumila, U. pumila £U. wilsoniana, and U. wilsoniana £ U. minor. Theother crosses have brought to only one resistant andvaluable clone each (Table 3).
Adaptation trials
In Fig. 4 the mean height and diameter incrementsof the best clones are reported. The data concern twoadaptation trial Welds set up in sites with diVerentclimates. The mean current increment in height anddiameter in site A (Mountain region) were 24.5 cmand 0.4, respectively. In site B (Coastal area) were85.5 and 1.2 cm, respectively. The data show that onaverage the growth was better under the coastal cli-mate, although a few selections found better condi-
Fig. 2 Number of elm clones obtained (white bars) and number of clones considered resistant (less than 25% of dieback) to Dutch elmdisease at the second screening (black bars) for each cross between diVerent species
0102030405060708090
100
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52 Euphytica (2008) 163:45–56
tions in the mountainous area as, for example FL385or FL390 who showed an increment of 75 cm, sig-niWcantly higher than the average, while in site Btheir increment is not diVerent from the generalaverage. Some clones had no increment in the lastyear of the trial and some other suVered the death ofall the ramets—as clone FL294 in site A and FL385in site B.
An unexpected interesting outcome of the adapta-tion trials was the survey of damages that local patho-gens of minor importance on native elm species mayproduce on elm species introduced from abroad andon related hybrids. The most exciting Wnding was theobservation of heavy damages attributable to ElmYellows (EY) on some Asian species and theirhybrids. As far as 1985, EY were considered to be adisease just for North America, where they kill Ulmusamericana. Yet in Europe, EY is tolerated by the pop-ulations of native elms, with only a few individualsshowing symptoms of yellowing, witches’ brooms,growth retardation, or a general decline (Mittemper-gher 2000; Mittempergher and Santini 2004). Anotherparasite taken into consideration in our adaptabilityplots has been the insect Elm Leaf Beatle (ELB)(Xantogalerucha luteola Mueller), which does peri-odical pullulations on U. minor. We found that theChinese U. laciniata (Trautv.) Mayr is so susceptibleto ELB that it is diYcult to rise it in central Italy with-out chemical control, whereas the Chinese elmsU. parvifolia and U. wilsoniana are scarcely dam-aged. A rating of susceptibility to Elm Yellows and toElm Leaf Beatle for a number of elm species was
published by one of us (Mittempergher 2000). TheseWndings should be taken into account when preparingand carrying out breeding projects.
Fig. 3 Resistant hybrid elm clones ranked for increasing symptoms of Dutch elm disease expressed as percent of dieback
0
10
20
30
40
50
60
70
80
90
100
FL
193
FL
405
FL
338
F
L 3
70F
L 5
06F
L 3
90F
L 2
79F
L 3
35
FL
007
FL
385
FL
418
FL
371
FL
047
F
L 3
02F
L 5
36F
L 1
93F
L 3
65F
L 3
24
FL
371
FL
301
FL
292
FL
327
FL
356
F
L 0
30F
L 0
45F
L 0
99F
L 1
00F
L 3
89
FL
376
FL
403
FL
416
FL
339
FL
034
FL
387
F
L 2
94F
L 3
37F
L 0
42
FL
041
FL
033
FL
099
FL
368
FL
315
FL
690
FL
386
FL
333
F
L 0
68F
L 1
89F
L 0
28F
L 1
09F
L 3
62F
L 1
98F
L 0
33F
L 3
67F
L 0
46F
L 0
27F
L 3
88F
L 1
88F
L 1
37F
L 0
31F
L 1
94F
L 0
44F
L 1
33F
L 0
78
clones
per
cen
t o
f sy
mp
tom
s
% defoliation
% dieback
Fig. 4 Height (a) and diameter (b) current increments of hybridelm clones considered resistant to Dutch elm disease, grown intwo sites characterised by contrasting climates: A typical Med-iterranean and B mountain Mediterranean climate
0
20
40
60
80
100
120
140
160
FL
033
FL
189
FL
294
FL
301
FL
302
FL
315
FL
337
FL
339
FL
385
FL
390
FL
416
FL
418
FL
506
clone
hei
gh
t in
crem
ent
(cm
) incr H05-06 A
incr H05-06 B
a
0
0.5
1
1.5
2
2.5
FL
033
FL
189
FL
294
FL
301
FL
302
FL
315
FL
337
FL
339
FL
385
FL
390
FL
416
FL
418
FL
506
clone
dia
met
er in
crem
ent
(cm
) incr D05-6 A
incr D05-6 B
b
123
Euphytica (2008) 163:45–56 53
Table 3 Elm clones considered resistant after two steps of artiWcial inoculations and evaluated as adapted to Mediterranean climateon the basis of adaptation trials
Clone Defoliation STD Dieback STD Score Cross
FL 333 5.00 2.50 11.11 4.90 ** ca £ ch
FL 356 8.88 8.58 4.44 3.00 ** ca £ wi
FL 025 20.46 10.07 13.46 12.06 * el £ pu
FL 506 8.50 5.79 0.16 0.41 *** ho £ ch
FL 041 22.50 7.55 8.00 2.58 * ho £ pu
FL 028 27.50 13.39 12.00 6.75 * ho £ pu
FL 044 43.50 6.26 21.00 6.99 * ho £ pu
FL 030 10.55 6.82 4.44 3.00 ** ho £ pu
FL 045 11.50 6.26 4.50 2.84 ** ho £ pu
FL 099 19.50 7.68 5.50 3.00 ** ho £ pu
FL 034 22.50 5.40 7.00 2.58 ** ho £ pu
FL 042 18.00 9.19 8.00 4.22 ** ho £ pu
FL 046 27.50 12.30 14.00 8.10 ** ho £ pu
FL 031 37.50 26.90 20.50 29.01 ** ho £ pu
FL 194 41.00 5.68 21.00 5.16 ** ho £ pu
FL 100 6.50 5.80 6.00 2.11 *** ho £ pu
FL 033 25.50 16.06 8.00 2.58 *** ho £ pu
FL 027 23.89 6.97 14.40 5.83 *** ho £ pu
FL 193 8.33 2.50 3.33 2.50 ** ho £ wi
FL 189 19.00 10.22 11.50 4.12 *** ho £ wi
FL 198 25.50 4.08 12.20 2.18 *** ho £ wi
FL 315 19.50 9.56 9.00 6.58 *** ja op
FL 389 9.50 9.56 6.00 5.60 * ja £ pu
FL 387 18.00 14.76 7.50 7.30 * ja £ pu
FL 368 11.50 17.01 9.00 10.00 * ja £ pu
FL 388 20.00 13.54 16.00 17.00 * ja £ pu
FL 365 12.77 12.91 3.66 4.06 ** ja £ pu
FL 324 2.50 3.78 3.75 2.30 ** ja £ pu
FL 327 7.22 12.78 4.44 3.90 ** ja £ pu
FL 362 38.75 25.59 12.00 8.04 ** ja £ pu
FL 367 21.00 12.65 13.00 7.90 ** ja £ pu
FL 390 3.00 2.58 1.00 0.60 *** ja £ pu
FL 302 6.10 5.46 3.00 2.64 *** ja £ pu
FL 292 9.00 7.75 4.00 2.11 *** ja £ pu
FL 294 25.50 13.01 7.50 3.54 *** ja £ pu
FL 386 33.88 19.92 9.89 7.00 *** ja £ pu
FL 301 8.90 5.46 4.00 3.33 **** ja £ pu
FL 536 8.89 5.16 3.22 3.85 *** pa op
FL 418 6.38 3.54 2.13 3.60 *** pa £ la
FL 403 20.00 12.91 6.40 4.06 ** pa £ pu
FL 405 0.00 0.00 0.00 0.00 *** pa £ pu
FL 416 10.71 8.59 6.57 9.34 *** pa £ pu
FL 279 2.20 3.63 2.00 2.50 ** pa £ wi
123
54 Euphytica (2008) 163:45–56
These results re-enforced the idea that adaptationtrials, even if they need a long time to give results,and they are space- and money-consuming, have afundamental role for successfully release the selectedclones on the market. Moreover a clear example ofgenotype £ environment interaction was revealed, sothat each selection needs its own optimum to expressall its potential.
Conclusions
The breeding program performed in Italy has so farbrought to 4 resistant (1 hybrid from pure species and3 obtained by crosses of hybrids) and adapted elmclones (Santini et al. 2002, 2007). Although this is asatisfying results, the work is not yet Wnished sinceseveral other clones have shown valuable charactersso far, and are in the process of being released. In thefuture the perspective is to obtain a wide range ofDED resistant clones with diVerent parentage in order
to further safeguard against the appearance of newand even more aggressive strains of the pathogen, asit occurred in the 1970s when the O. novo-ulmiappeared in Europe, or against diVerent unpredictablerisks.
Moreover, the strategy adopted for elm geneticimprovement, i.e. incorporation, had produced a newpopulation that have high proportion of unique,exotic-derived alleles that indeed broaden the elmgenetic base, so that the variance will result enhanced(Simmonds 1993). The gain in genetic richness andvariability should allow to encompass the strong bot-tleneck that, for the use of exotic species, even ifhybridised with endogenous germplasm, is repre-sented by adaptation to new environments.
The introduction of non-native species oftenremove geographical barriers to mating and lead tohybridisation and introgression that would not occurnaturally (Thymer and SimberloV 1996). Suchhybridisation could be a threat for biodiversity atlocal or regional level, leading to irreversible
Table 3 continued
Defoliation is the mean percent of wilting symptoms assessed after one month from the inoculation date. Dieback is the mean percentof dieback symptoms assessed one year after the inoculation. STD = standard deviation of each parameter. The number of stars in theScore column means the level of satisfaction of the features taken into account, from one mark = clone with barely enough feature tobe acceptable, to four marks = clone that accomplish all the requested characters: resistance, adaptation, leaf colour and shape, trunkand crown. Cross: Species codes ca = U. minor; ch = U. chenmoui; wi = U. wilsoniana; pu = U. pumila; ho = U. £ hollandica;pa = U. parvifolia; el = U. elliptica; la = U. laevis; ja = U. japonica; op = open pollinated.
Clone Defoliation STD Dieback STD Score Cross
FL 007 7.20 11.76 2.00 2.50 ** pu £ ca
FL 109 29.00 8.10 12.00 4.83 ** pu £ ca
FL 078 60.00 12.47 24.50 6.43 ** pu £ ca
FL 068 22.22 14.60 11.11 9.93 *** pu £ ca
FL 690 11.25 13.33 9.10 9.17 *** pu £ ja
FL 385 6.50 5.30 2.10 2.18 *** pu £ pa
FL 376 14.17 16.35 6.33 7.44 ** pu £ wi
FL 370 1.00 0.00 0.11 0.33 *** pu £ wi
FL 371 3.33 4.33 2.22 3.60 **** pu £ wi
FL 137 29.50 8.64 18.50 13.34 * wi £ ca
FL 133 54.00 19.55 24.00 8.10 * wi £ ca
FL 188 38.50 13.95 17.00 6.32 ** wi £ ca
FL 338 0.62 1.77 0.00 0.00 ** wi £ pu
FL 335 5.00 7.45 2.00 2.60 ** wi £ pu
FL 047 1.90 2.59 3.00 2.67 *** wi £ pu
FL 339 17.00 9.78 7.00 4.20 *** wi £ pu
FL 337 22.00 2.42 7.70 1.03 **** wi £ pu
123
Euphytica (2008) 163:45–56 55
changes to native species and hence causing massiveecological and economic damage. The problem hasalready arisen back in the 1930’s when Siberian elmwas introduced to Italy in order to limit the lossesdue to the Wrst DED epidemic. This species naturallyhybridised with the native Weld elm (Goodall-Copes-take et al. 2005; Cogolludo-Agustiân et al. 2000;Mittempergher and La Porta 1991) giving concernfor the genetic integrity of the native species (Collinet al. 2000). On the other hand, hybridisation is afrequent and important component of plant evolu-tion and speciation (Riesenberg and Ellstrand 1993),and it was also suggested that more than 70% ofplant species may be descended from hybrids (Grant1981).
Acknowledgements The C.N.R. elm collections could nothave been constituted without the help and the sharing spirit ofmany colleagues from all over the world, to whom our warmestthanks are devoted. In particular we wish to thank Hans M.Heybroek, Eugene B. Smalley, Raymond P. Guries, Alden M.Townsend, and Frank S. Santamour Jr.. The unique technicalassistance of Mr. Fabio Ferrini, Mr. Alberto Fagnani and Mr.Abdellah Dahmani made possible the realisation of this 30-year-long work. Authors wish to thank Dr. Stefano Vagniluca and Dr.Luciano Sammarone of the Corpo forestale dello Stato UTBFollonica (GR) and UTB Castel di Sangro (AQ) respectively,for having provided and taken care of the experimental Welds.Authors wish to thank Hans M. Heybroek and Eric Collin for thecritical review of the paper, for their friendship and for all theuseful suggestions along these years.
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