Sri Rahayu et. al.·Silvae Genetica (2009) 58-1/2, 62-68

Appendix B. – Full correlation matrix of yearly cumulative cone yield (CCY), with standard errors in parentheses from the pair-wise analysis. Genetic and phenotypic correlations are given in the upper and lower diagonals, respectively.

Responses of moluccana seedlings of Different Seed Sources to Inoculation With tepperianum

By SRI RAHAYU1),2),*), NOR AINI AB. SHUKOR2), LEE SU SEE3) and GHIZAN SALEH4)

(Received 7th January 2008)

Abstract disease causes severe damage at all developmental (batai) is one of the valuable stages of the from the nursery stage to mature multipurpose tree species for forest plantations, particu- trees in the field and includes the development of choco- larly in and . Gall disease late brown, cauliflower-like or whip-like galls on the caused by (Sacc.) is one of stem, branch, petiole, shoot and pod. Different seed the most destructive diseases in Batai plantations. The sources may respond differently to gall rust . Thus, the responses of F. moluccana seedlings from 6 selected seed sources to gall rust disease caused by U. tepperianum, were evaluated, at the Brumas Estate, 1 ) Department of Silviculture, Faculty of Forestry, Gadjah Mada Malaysia, in terms of gall rust disease severity, mortali- University, Bulaksumur, Yogyakarta 55281, Indonesia. ty, and disease infection rate of the seedlings. Based on 2 ) Institute of Tropical Forestry and Forest Products, University disease severity, infection rate and cumulative mortality Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia. due to gall rust disease, the wamena was found to be the 3 ) Forest Research Institute Malaysia, Kepong, 52109, Selangor, best seed source in relation to gall rust disease resis- Malaysia. tance. 4) Department of Crop Science, University Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia. Key words: Falcataria moluccana, seed sources, Uromycladium *) Corrersponding author. E-mail: [email protected] tepperianum, artificial inoculation, resistance.

62 Silvae Genetica 58, 1–2 (2009)

DOI:10.1515/sg-2009-0008 edited by Thünen Institute of Forest Genetics Sri Rahayu et. al.·Silvae Genetica (2009) 58-1/2, 62-68

Introduction found to infect old decurrens trees on the Ijen Falcataria moluccana ((Miq.) Barneby and J.W. plateau in East of , at about 2100 m above sea level Grimes (batai)), of the family (alt. Legumi- (SRI RAHAYU, 2007, unpublished data). nosae), is a fast growing tree frequently planted in plan- In the entire life cycle of the fungus, U. tepperianum tations forests in the humid tropics (WAGNER et al., (Sacc.) McAlp. it produces only one single spore type, 1999). This species is best known as a pulp crop (NAS, namely teliospore. Under nursery conditions, teliospores 1979), but is also used for fiber and particleboard, pack- of the rust fungus cannot themselves infect the host, ing cases, boxes, matches, chopsticks, light furniture, they first have to germinate to produce basidiospores lightweight veneer and plywood and composite products (DICK, 1985; RAHAYU, 2007). The Uromycladium fungi (CABI, 2000). Since the wood is fairly easy to cut, are autoecious, only requiring one host to complete their F. moluccana is also suitable for wooden shoes, musical life cycle (MORRIS, 1987). instruments, toys and novelties and also general turn- The disease causes severe damage at all developmen- ery (PEH and KHOO, 1984). The species has also been tal stages of the plant from the nursery stage to mature used as shade trees to coffee, tea and cacao and trees in the field and this involves the development of cattle because of its favorable thin crown. According to chocolate brown, cauliflower-like or whip-like galls on SCHUBERT (1985), it is also used as windbreakers for the stem, branch, petiole, shoot and pod. Affected plant banana plants in . Batai is also grown as an parts and severely infected F. moluccana trees die pre- ornamental tree, although it seldom survives for more maturely (CRISTOVAO and OLD, 2003). On the other hand, than 50 years (BUDELMAN, 1989). due to its host specificity, the gall-forming rust fungus F. moluccana occurs naturally in Indonesia (Moluccas U. tepperianum from was selected as a suit- and Irian Jaya islands), Papua , New able biological agent for controlling an invasive tree Britain and the , ranging from 10° S to in the Western Cape Province of Africa 30° N (WAGNER et al., 1999; RICHTER and DALLWITZ, and was able to decrease tree density by at least 80% at 2000). It is an exotic tree species planted in Brunei, all sites in 1995 (MORRIS, 1997). Experience with rust Cambodia, Cameroon, Cook Islands, Fiji, French Poly- diseases of other tree genera elsewhere, shows that nesia, Japan, Kiribati, Laos, Malaysia, Marshall there may be major differences in resistance by species, Islands, Myanmar, , Norfolk Island, provenance, family and seed source levels. However, the , , Thailand, Tonga, United States of impact of the disease has not previously been quantita- America, Vanuatu and Vietnam (WAC, 2005). Gall rust tively assessed in different seed sources of F. moluccana disease caused by Uromycladium tepperianum (Sacc.) seedlings. In this paper the responses F. moluccana (RAHAYU et al., 2005) is one of the most destructive dis- seedlings, from different seed sources, to gall rust dis- eases in these plantations in some parts of the South ease caused by U. tepperianum, encompassing gall rust East Asian region, such as in the Philippines (Braza, disease severity, mortality and infection rate of the 1997), Sabah in Malaysia (LEE, 2004) and some islands seedlings were evaluated in Tawau, Sabah, Malaysia. in Indonesia (RAHAYU, 2007). U. tepperianum is a micro- cyclic rust that infects more than a hundred host Materials and Methods species, U. tepperianum also infects Acacia spp.. in Aus- tralia and New Zealand (OLD et al., 2000), but it has Seedlings material never been reported to have infected Acacia spp. in F. moluccana seedlings originating from six seed Malaysia. However, in Indonesia, the fungus had been sources were evaluated. Detailed information of each

Table 1. – List of F. moluccana seed sources used to screen for resistance of gall rust.

Note: - = no data

63

DOI:10.1515/sg-2009-0008 edited by Thünen Institute of Forest Genetics Sri Rahayu et. al.·Silvae Genetica (2009) 58-1/2, 62-68

seed source is presented in Table 1. The seed lot for each Severity, mortality and infection rate number comes from one tree with no outcrossing Symptoms exhibited by gall rust on seedling vary in between each individual tree. The seeds were initially different plant tissue and could appear on the shoot, leaf treated with boiling water at 90°C for five minutes, fol- stalk and stem. The scores for gall rust disease were lowed by soaking them in tap water for 12 hours before based on estimations made on the stem since earlier they were sown. The seeds were sown directly in 8 x 8 x findings indicated that the stem is the most susceptible 12 cm polyethylene bags containing sand and topsoil in to it (RAHAYU et al., 2006). In addition, based on the size ratios of 3:7 (v/v). The seedlings were maintained in the of gall rust and seedling malformation due to fungus nursery for 6 weeks. At 6 weeks seedlings with height infection, gall rust indices scores were grouped into four ranging from 7 to 11 cm, were moved to the field site. categories as presented in Table 2. A preliminary study showed that full symptoms of gall rust developed within Fungal material 6 to 7 days after inoculation (DAI), therefore the scores General rust fungus easily forms pathogenic races. for gall rust disease was initially recorded every 10 days However, there is no information on the pathogenic for 7, 17, 27 and 37 days. races of U. tepperianum found on F. moluccana up to Based on the index score for gall rust symptoms, gall now. Spores for inoculum were obtained from galls of rust disease severity (DS) was calculated using modified infected F. moluccana seedlings originating from a sin- Chester’s formula (1959) as follows: gle seed source in the nursery. Accordingly the inoculum

is expected to be from one race. Crude scrapings of galls Disease Severity (DS) = {[(n0 x z0) + (n1 x z1) + ... + were made to collect teliospores which were subsequent- (n3 x z3)] / (N X Z)} x 100% ly suspended in distilled water containing Tween 20 where: (0.2 ml/l). DS = Disease Severity n , n , n , n = number of trees with index score 0, 1, 2 Inoculation 0 1 2 3 and 3 Six week-old healthy seedlings were used as experi- z , z , z , z = index score 0, 1, 2, 3. mental units. Inoculum density of the first, second and 0 1 2 3 N = Total number of trees in one plot (4), third inoculation were 23.4 x 106, 3.7 x 106 and 14 x 106 Z = the highest score (3) teliospores/ml, respectively. For each inoculation, a total of 5 ml inoculum was applied onto each seedling, with The infection rate of gall rust (r) per unit time (t) was 3 ml dripped on the stems from the shoot tip using a calculated using the following formula as suggested by measured pipette, and 2 ml sprayed on the entire KURT (2002). seedling using a spray bottle. Control seedlings were r = 1/ (t2 – t1) x [(ln(x2/1-x2)) – (ln(x1/1 – x1))] treated using the same procedure using distilled water as inoculum. All seedlings of this study were maintained where: in the nursery and watered twice a day, up to field t1 and t2 = the dates on which the disease severity capacity. measurements were made

x1 and x2 = disease severity (%) recorded on these Experimental Design dates A Randomized Complete Block Design (RCBD) with 3 Based on the assumptions about gall rust disease blocks, 7 replications in each block and 4 unit samples resistance of F. moluccana seedlings of polygenic inheri- in each replication were set up separately for the gall tance, the typical mortality curve can be used to differ- rust inoculation and the control. In order to prevent con- entiate seed sources with potential resistance. Gall rust tamination of control seedlings by teliospores from the symptoms with a score of 3, noted as dead seedlings, inoculated seedlings, the distance between the inoculat- was calculated for 7 to 37 DAI. The percentage of dead ed and control plots was at least 100 m. The plant part seedlings at each observation period was considered as observed was the stem of the seedlings. The inoculated the cumulative seedling mortality. Based on gall rust treatment consisted of 3 blocks x 7 replications x 4 unit disease severity and mortality, the response of F. moluc- samples x 6 seed sources = 504 seedlings as unit obser- cana seedlings to gall rust disease was grouped into four vations, as well as for the control treatment. categories as presented in Table 3.

Table 2. – Index score for gall rust symptoms on F. mollucana seedlings under shade trees.

64

DOI:10.1515/sg-2009-0008 edited by Thünen Institute of Forest Genetics Sri Rahayu et. al.·Silvae Genetica (2009) 58-1/2, 62-68

Table 3. – Resistance category of F. moluccana seedlings to gall rust dis- ease caused by Uromycladium tepperianum based on gall rust severity and percent mortality.

Table 4. – Key-outs for ANOVA on gall rust disease severity and mortality of F. moluccana seedlings.

According to STUBBS et al. (1986) and ZADOKS and Results SCHEIN (1979), the logarithmic function ((ln(x/(1 – x)), Severity, Mortality and Infection rate which is termed as the logit of x, the development of gall rust disease symptom on each seed source can be plotted Summary of the results of the analysis of variance for as a straight line as shown in the following : disease severity, showed that effect of blocks and also seed sources were not significant. However interaction y = a + bx between blocks and seed sources was significantly from where: 7 DAI to 37 DAI. This finding indicates that cumulative variation within blocks and seed sources was large and y= logit of the amount of gall rust infection that subsequent block conditions particularly the micro- a= gall rust severity at first observation climate surrounding the individual seedlings have a b= the slope or the disease rate (r) great effect on gall rust disease severity. The mean per- x= the number of days after the first observation. formances of the F. moluccana seedlings from the six seed sources for gall rust disease severity are presented Statistical Analysis in Fig. 1. The wamena seed source was found to be the The following linear additive model was employed to only seed source which showed resistant (R). estimate the components of variance for screening of seed sources: τ β τβ ε Yij = µ + i + j + ij + ij where:

Yij = Disease severity, cumulative mortality, height, or relative growth rate mean of 4 seedlings of seed sources i in block j. µ= The population mean τ i = The random variable effect for block j, j = 1, 2, 3 β j = The fixed effect for seed source, i = 1 s/d six i.e. Wamena, Walang Gintang, Morotai, Kediri, and 2S/75 seed sources τβ ij = The random interaction of seed sources by block ε σ2 ij = The error term ~NID (0, e; where i = 1, 2, 3 block; j = 1, 2, 3, ... , six seed sources) τ σ2 β β ε σ2 Assumptions: i ~ NI (0, t), j ~ NI (0, b), ij ~ NI (0, ) Data were subjected to two way analyses of variance (ANOVA) which was carried out separately for the inoc- Figure 1. – Mean performance of gall rust disease severity in ulated and control seedlings. The ANOVA table key-outs six different seed sources of Falcataria moluccana, inoculated with the expected means squares (EMS) used for each with U. tepperianum teliospores, at 7, 17, 27, and 37 DAI, with different categories: S = susceptible (≥75%–100%), M = moder- treatment are as given in Table 4. The analysis utilized ate (≥25%–74%), R = resistant (0%–25%), vertical bars repre- the PROC GLM test to measure the significance of these sent standard deviation; bars in the same range are not signifi- random effects (SAS INSTITUTE, 2004). cantly different at P ≤ 0.05.

65

DOI:10.1515/sg-2009-0008 edited by Thünen Institute of Forest Genetics Sri Rahayu et. al.·Silvae Genetica (2009) 58-1/2, 62-68

Discussion Gall rust disease caused by Uromycladium tepperi- anum is a major threat to F. moluccana plantations in Sabah Malaysia. Affected seedlings usually lose their leaves and become stunted, eventually dying with dis- ease incidence in the nurseries at 90 to 100 percent (DE GUZMAN et al., 1991). Further, 20 days infection of gall rust fungus, separately on the stem, shoot and leaf stalk, on two month old seedlings caused 82%, 70% and 73% mortality, respectively (RAHAYU et al., 2006). This may relate to the abnormal physiological process of the seedlings with galls. Abnormalities are generally caused by either hypertrophy or hyperplasia or both. Hypertro- phy is defined as an abnormal over-development due to an increase in cell size whereas hyperplasia is the increase in cell number due to the disease producing Figure 2. – Mean performance of gall rust disease mortality in agent (TOOTHILL, 1984). In addition, mycelium of the six different seed sources of Falcataria moluccana, inoculated rust fungus is typically perennial in the bark or wood of with U. tepperianum teliospores, at 7, 17, 27, and 37 DAI, with the host tree. This resident mycelium causes various different categories: S = susceptible (>75–100%), M = moder- host malformations, including bending of stem or shoot ≤ ate (>25 – 75), R = resistant (0–25%), vertical bars represent due to hypertrophy or hyperplasia on the meristematic standard deviation; bars in the same range are not significant- ly different at P ≤ 0.05. cells (RAHAYU, 2007). Further gall formation also leads to stoppage of water movement in the xylem (EDMONDS et al., 2000). Generally, resistance to rust fungus is polygenic, and the characteristics of the mortality can be used to differ- entiate the potentially useful levels of resistance from susceptible seed sources (STINE, 1999). However, this study found no significant differences in gall rust dis- ease severity and mortality in seedlings from the 6 F. moluccana seed sources treated. This evidence may be related to the infection mode of gall rust fungus caused by U. tepperianum on F. moluccana. According to RAHAYU (2007), basidiospores of U. tepperianum are capable of direct penetration through the epidermis by formation of a penetration peg. This is followed by an enzymatic process infection and invasion of the gall rust fungus in the host cells. Thus, all the seedlings from the six seed sources were infected by the rust fungus, although, subsequent fungus development in the cell hosts differs for each seed source. In addition, given that Figure 3. – Logit plot of the gall rust infection rate of F. moluc- the cell wall is the first line of defense against rust cana seedlings from six seed sources at 7 to 37 days after inoc- infection, it is not surprising that the plants evolve ulation, vertical bars represent standard deviation; bars in the means to perceive and respond defensively forming galls same range are not significantly different at P ≤ 0.05. (MELLERSH and HEATH, 2001). Since the stems of the seedlings of all the seed sources became infected in this experiment, it is apparent that the seedlings in this The analysis of variance for cumulative mortality study lack mechanisms of resistance that eliminate showed that effect of blocks, seed sources and interac- infection. Consequently, it is unlikely that seedlings tion between blocks and seed sources were not signifi- with a high degree of resistance can be found, meaning cant. However, cumulative mortality of seedlings origi- that no seedlings will be free from U. tepperianum infec- nating from the wamena seed source showed resistance tion. from 7 to 37 DAI, while the others showed moderate Based on resistance category, only the wamena seed resistance beyond 37 DAI (Fig. 2). source showed resistance up to 37 DAI, while the other Gall rust infection rate (r) from 1 to 37 DAI presented seed sources displayed moderate resistance. In addition, as the logit plot of gall rust infection (Fig. 3), shows that the gall rust infection rate of the seedlings from the seedlings from the wamena seed source had the lowest wamena seed source at 1 to 37 DAI was found to be the infection rate, significantly different from the others. lowest and significantly different from the other seed Seedlings from the other seed sources showed higher sources. In their natural distribution, F. moluccana infection rates, with seedlings from the Maluku seed trees in Wamena grow in groups around the Wamena source displaying the highest rate of infection. village at about 2000 m above sea level. In that area,

66

DOI:10.1515/sg-2009-0008 edited by Thünen Institute of Forest Genetics Sri Rahayu et. al.·Silvae Genetica (2009) 58-1/2, 62-68

fogs are frequent and when clouds blow through the BUDELMAN, A. (1989): falcataria-Southeast foliage, nutrients may be ‘fog-stripped’ leaching them Asia’s Growth Champion. Royal Tropical Institute. out unless the plants have protective impermeable cuti- Amsterdam, The Netherlands. cles. Fog is one of the most significant factors supporting CABI (2000): The Forestry Compendium – A Silviculture gall rust disease development in F. moluccana (RAHAYU, Reference. Module 1. Commonwealth Agricultural 2007). Due to the seed source from Wamena having Bureau International, Wallingford, U.K. adapted to foggy conditions, their anatomical and mor- CHESTER, K. S. (1959): How sick is the plant. J. G. H phological properties such as impermeable cuticles and HORSFALL and A. DIAMOND eds., Plant Pathology Vol: 1. intracellular modification are thus more resistant to gall Academic Press, Inc, New York. rust fungus infections. In addition, based on histopatho- CRISTOVAO, C. S. and K. M. OLD (2003): A rust epidemic of the coffee shade tree (Paraserianthes falcataria) in East logical observations, there was also evidence of hyper- Timor. ACIAR Proceedings No. 13. 139–145. sensitive reaction inside infected stem cells of seedlings DICK, M. (1985): Uromycladium rusts of Acacia. New from the wamena seed source. Cells, particularly xylem Zealand Forest Service, Forest Pathology in New cells with houstorial cells die swiftly as in the seedlings Zealand No. 15. 8 p. cells from wamena, while in other seed sources, houstor- DE GUZMAN, E. D., E. P. MILITANTE and R. LUCERO (1991): ial cells developed rapidly and occupied all the xylem Forest nursery diseases and insects in the Philippines. cells. According to GOODMAN and NOVACKY (1994) and Pages 101-104. In: SUTHERLAND, J. R. and GLOVER, S. LEVINE et al. (1994), hypersensitivity is an effective G., eds. Proceedings of the first meeting of IUFRO defence mechanism against biotrophs and non-biotrophs working party S2.07-09 (diseases and insects in forest alike because of the accompanying upregulation of a nurseries). Victoria, British Columbia, Canada, August multitude of defense genes that produce a highly 22–30, 1990. Forestry Canada, Pacific Forestry Centre, antimicrobial environment in and around the dead cells. Information Report BC-X-331. Further intensive research on the hypersensitive reac- EDMONDS, R. L., J. K. AGEE and I. G. ROBERT (2000): For- tions of some provenances, progenies or seed sources of est Health and Protection. Mc Graw Hill, New York. F. moluccana is needed in order to ascertain the materi- GOODMAN, R. N. and H. J. NOVACKY (1994): The hypersen- als resistant to gall rust fungus. sitive reaction in plants to pathogens. St. Paul: APS Press. There is a correlation between the result of artificial KURT, S. (2002): Screening of wheat cultivars for resis- inoculation and the performance of the same materials tance to stripe rust and leaf blotch in Turkey. Crop Pro- in the field (POWERS et al., 1982). Highly susceptible tection 21: 495–500. materials in the greenhouse were also highly susceptible LEE, S. S. (2004): Diseases and potential threats to Acacia in field plantings. The problem usually involves families mangium plantation in Malaysia. Unasylva 217. (55): rated as moderate in resistance. This seems to be partic- 31–35. ularly true for loblolly pine. It was found that some LEVINE, A., R. TENHAKEN, R. DIXON and C. LAMB (1994): loblolly pine families which appeared to be relatively H2O2 from the oxidative burst orchestrates the plant susceptible to fusiform rust in inoculation tests were hypersensitive disease resistance response. Cell 79: moderately resistant under field conditions. Neverthe- 583–593. less the reverse was not found to be true. Those condi- MALLERSH, D. G. and M. C. HEATH (2001): Plasma mem- tions may be due to the seedlings in the green house brane-cell wall adhesion is required for expression of being subjected to strong treatments in order to stimu- plant defense responses during fungal penetration. late the disease. On the other hand, under field condi- American Society of Plant Pathologist. Plant Cell. 13: tions, varying factors occur which either stimulate or 413–424. inhibit disease development. Thus, in the case of gall MORRIS, M. J. (1997): Impact of the gall-forming rust fun- rust disease screening, there is a good chance that the gus Uromycladium tepperianum on the invasive tree seedlings from seed sources which demonstrated resis- Acacia saligna in . Biological Control. 10: 75–82. tance (wamena) in artificial inoculation may also show N.A.S. (1979): Tropical legumes: Resources for the Future. resistance in the field. Since wamena was found to be National Academy Press. Washington, DC. 15 pp. the most promising resistant seed source in this test, PEH, T. B. and K. C. KHOO (1984): Timber properties of further tests should be done with regards to its resis- Acacia mangium, Gmelina arborea, Paraserianthes fal- tance to gall rust disease in the field. cataria and their utilization aspects. Malaysian Forester 47: 285–303. Conclusions POWERS, H. R. JR., S. D. HUBBARTD and R. L. ANDERSON (1982): Testing for resistance to fusiform rust of Pine. Based on artificial inoculation under field conditions Pages 427–434. In: HEYBROEK, H. M., STEPHAN, B. R. on F. moluccana seedlings, wamena was found to be the and VON WISSENBERG, K. eds. Resistance to disease and best seed source in relation to gall rust disease resis- pests in forest trees. Proccedings of the third interna- tance. Further tests should be done with regards to its tional workshop on the genetics of host-parasite interac- resistance to gall rust disease in the field. tion in forestry, Wageningen, the Netherlands, 14–21 September 1980: Pudoc. RAHAYU, S., S. S. LEE and A. S. NOR AINI (2005): Gall rust References disease in Falcataria moluccana (Miq) Barneby & BRAZA, R. D. (1997): Gall rust disease of Paraserianthes Grimes at Brumas, Tawau-Sabah. Pages 288–289. In: falcataria in the Philippines. Forest, Farm, and Com- SAHIBIN, A. R., RAMLAN, O., KEE, A. A. A. and NG, Y. F. munity Tree Research Reports 1997. 2: 61–62. eds. Proceeding of Second regional symposium on envi-

67

DOI:10.1515/sg-2009-0008 edited by Thünen Institute of Forest Genetics Pollegioni et. al.·Silvae Genetica (2009) 58-1/2, 68-78

ronment and natural resources, 22–23 March 2005. STINE, R. A. (1999): Reducing the impact of blister rust on UKM and Ministry of Natural Resources and Environ- white pine in Minnesota. Principle investigator. Final mental, Malaysia. Report. Minnesota. 28 pp. RAHAYU, S., S. S. LEE, A. S. NOR AINI, S. GIZAN and S. S. STUBBS, R. W., J. M. PRESCOTT, E. E. SAARI and H. J. AHMAD (2006): Infection of Falcataria moluccana (Miq.) DUBIN (1986): Cereal Disease Methodology Manual. Barneby & Grimes seedling by gall rust fungus Uromy- CIMMYT, Mexico. 46 pp. cladium spp. is associated with a reduction in growth TOOTHILL, E. (1984): Dictionary of Botany. Penguin. Great and survival. Pages 243–247. Proceeding of Internation- Britain. al Post Graduate Student Conference. Penang: Univer- WAC (World Agro forestry Centre). (2005): Agro forestry sity Science Malaysia (USM), Malaysia. Database. www.worldagroforestry.org/Sites/TreeDBS/ RAHAYU, S. (2007): Gall rust disease of Falcataria moluc- Aft/Print.cfm?SpID=171. Accessed on 12 February 2005. cana in Sabah, Malaysia PhD. Thesis. Universiti Putra WAGNER, W. L., D. R. HERBST and S. H. SOHMER (1999): Malaysia, Malaysia. Manual of the Flowering Plants of Hawaii. 2 vols. Bish- SCHUBERT, T. R. (1985): Preliminary results of eucalyp- op Museum Special Publication 83, University of tus/legume mixtures in Hawaii. NFTRR 3: 65–66. Hawaii and Bishop Press, Honolulu, Hawaii.

Characterization of Juglans nigra (L.), Juglans regia (L.) and Juglans x intermedia (Carr.) by SSR markers: a case study in Italy

By P. POLLEGIONI1), K. WOESTE3), A. MAJOR2), G. SCARASCIA MUGNOZZA1) and M. E. MALVOLTI1),*)

(Received 18th January 2008)

Abstract with two genome parts of J. nigra and one part of Juglans nigra and Juglans regia are economically J. regia was identified. The cytological analysis proved important species in Europe, Asia and North America. this triploid state showing 48 somatic chromosomes. The Natural hybrids between the two species, known as mother testing analysis of the 7 diploid hybrids by Juglans x intermedia (Carr), are valued for timber pro- exclusion method indicated one putative hybridogenic duction. We tested ten nuclear microsatellite markers to mother plants. The sequence analysis of amplified frag- (1) identify new J. x intermedia hybrids and character- ments confirmed the cross-species amplification of SSR. ize their parentage species J. regia and J. nigra (2) Inter-specific differences between alleles were due not detect J. nigra genotypes with a spontaneous crossing only to simple changes in the number of repeats but also ability with J. regia in a mixed Italian population. This to mutations in the flanking regions. study was also designed to confirm the transferability of Key words: Juglans, interspecific hybrids, microsatellites, ten black walnut SSR loci to Persian walnut All ten cross-species amplification, sequencing. microsatellites amplified in both species, producing frag- ments of variable size; eight (7.14%) were common, 68 (60.7%) amplified in J. nigra and 36 (32.1%) in J. regia Introduction only (private alleles). Indices of genetic diversity Juglans nigra (Eastern black walnut) and Juglans revealed high level of variability. The Principal Coordi- regia (common or Persian walnut) are highly economi- nate Analysis on the basis of total 112 alleles divided the cally important species in Europe, Asia and North Amer- total sample set into three main groups: J. nigra, J. regia and J. x intermedia hybrids. Performing the ica. The Persian walnut, an indigenous species in Eura- microsatellite fingerprinting, a triploid hybrid plant sia from the Balkans to southwest China, is cultivated throughout the temperate regions of the world for its high quality wood and edible nuts. Black walnut, native 1) C.N.R. Institute of Agro-environmental and Forest Biology, viale Marconi 2, 05010, Porano, Terni, Italy. E-mail: paola. to the Eastern part of North America, is a fast growing [email protected]; [email protected] species with a dark-colored wood used in the manufac- 2) Molecular Tassonomy Laboratory, Hungarian Natural History ture of furniture and other wood products. Beginning in Museum, Baross u. 13, 1088 Budapest, Hungary. E-mail: the 17th century, J. nigra was imported from the Eastern [email protected] and Central hardwood forests of the United States to 3) U.S.D.A. Forest Service, Hardwood Tree Improvement and the European continent for ornamental purposes, and Regeneration Center, Department of Forestry and Natural Resources, Purdue University, 715 West State Street, West subsequently for its rapid growth. In Italy the black Lafayette IN 47907-2061. E-mail: [email protected] walnut is usually found in private and public parks of *) Corresponding author: MARIA EMILIA MALVOLTI. Tel. 0039 Pianura Padana where is also used for reforestation and 0763374913. Fax 0039 0763374980. E-mail: [email protected] recovering degradated areas (FENAROLI, 1975). Both

68 Silvae Genetica 58, 1–2 (2009)

DOI:10.1515/sg-2009-0009 edited by Thünen Institute of Forest Genetics