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Gayle M. Volk, Christopher M. Richards, Adam D. Henk, Ann Reilley, Diane D. Miller and Philip L. Forsline

The wild apple species Malus sieversii (Ledeb.) M. Roem. is native to xeric regions and high mountain ranges of Kazakhstan, the Kyrgyz Republic, China, Tajikistan, Uzbekistan, and Turkmenistan ( Yan et al., 2008 ). With a wide range of edible

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D. Michael Glenn and Carole Bassett

population of Malus sieversii . Materials and Methods Irrigated ‘Empire’/‘M.7A'were planted at a density of 500 trees/ha in 1992 at the USDA/ARS Appalachian Fruit Research Station, Kearneysville, WV (lat. 39.3 N, long. 77.9 W, elevation 175 m). In all years

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Yi Tan, Baisha Li, Yi Wang, Ting Wu, Zhenhai Han and Xinzhong Zhang

cultivars, 96 commercial cultivars, 10 Malus sieversii accessions, 10 rootstocks, and 26 accessions of wild species (Supplemental Table 1). For the analysis of the inheritance of leaf regeneration ability, seventy-eight 11-year-old hybrid trees (Jonathan

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Tingting Sun, Tingting Pei, Zhijun Zhang, Mingjun Li, Linlin Huang, Cuiying Li, Xueyan Shi, Minghui Zhan, Xiaoyu Cao, Fengwang Ma and Changhai Liu

(CK = control, O = osmotic stress, OLP = combination of osmotic and low-P stresses, Ms = Malus sieversii , Mp = M. prunifolia , Mh = M. hupehensis ; Ms-R, Mp-R, and Mh-R stand for the root of different apple rootstocks; Ms-L, Mp-L, and Mh-L stand

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Stan C. Hokanson, Phil L. Forsline, James R. McFerson, Warren F. Lamboy, Herb S. Aldwinckle and Aimak D. Djangaliev

Malus sieversii, the main progenitor of domesticated apple, is native to areas in Central Asia. To better represent Malus wild germplasm in the USDA–ARS germplasm collections, maintained in Geneva, N.Y., a cooperative project was initiated with the Republic if Kazakhstan to collect and assess that country's wild populations of M. sieversii and to develop more secure in situ reserves to complement ex situ holdings in the United States and Kazakhstan. To date, four exploration trips to the region have included participants from the United States, Kazakhstan, Canada, New Zealand, and South Africa. Four Kazkh scientists have toured USDA–ARS sites, exchanged information, and collected germplasm in the United States greenhouse screens of 1600 have revealed potentially new sources of resistance to apple scab, cedar apple rust, and fire blight. An isozyme analysis of maternal half-sib families from four regions suggests the populations of M. sieversii collected represent a single panmictic population, with over 85% of total genetic variation due to differences among families. The most recent collections in 1995 were directed towards more ecologically diverse regions, including a site (Tarbagatai) at the most northern limit for M. sieversii equivalent to northern Minnesota in the United States. Some trees in this region produced fruit nearly 70 mm in diameter with excellent aroma, firmness, and color. This germplasm is being systematically characterized for horticultural traits, pest and disease resistance, and molecular markers.

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Philip L. Forsline, E.E Dickson and A.D. Djangaliev

The USDA National Plant Germplasm System (NPGS) sponsored a 1993 collection of wild Malus in Kazakhstan and Kyrgyzstan which followed a collection in 1989 from sites in Tajikistan and Uzbekistan. There is strong evidence that the domestic apple originated in the foothills of the Tian Shari mountains where Malus sieversii (Ldb.) M. Roem remains as a primary forest species. The goal of the recent expedition was to obtain additional genetic diversity of apple from some of the remote sites in that area with the assistance of the Kazakh hosts. While there, isolated pockets of other fruit in the wild (especially Vitis) were discovered and collected. Seed collections from the expedition are stored with the NPGS and seedling populations are being evaluated for valuable traits.

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D.K. Isutsa, I.A. Merwin and B.B. Brodie

Apple replant disease (ARD) is a serious problem in fruit production, and none of the major clonal rootstocks are resistant to ARD. We have screened Malus domestica clones and species accessions from the USDA Malus Germplasm Repository at Geneva, N.Y., including M. angustifolia-2375.03 (MA), M. coronaria-2966.01 (MC), M. fusca-3031.01 (MF), M. ioensis-3059.01 (MI), M. sieversii-3530.01 (MS), and M. kirghisorum-3578.01 (MK), for resistance to ARD and root-lesion nematodes (RLN, Pratylenchus spp.), in a composite soil collected from 11 New York orchards with known ARD. Plant dry mass and height, root necrosis, and nematode populations in different apple species and clones were compared after 60 days growth in steam-pasteurized (PS), RLN-inoculated (IS), and naturally infested field (FS) soils with 1200 RLN per 100 cm3. More severe stunting, reduced plant dry mass, and root necrosis occurred in FS seedlings compared with those in PS, but M. angustifolia seedlings were substantially more resistant or tolerant to RLN and ARD than the other species tested. Plant dry mass ranked MK>MS>MA>MI>MF>MC, and these differences were significant at the 5% level. RLN root populations were negatively correlated with plant dry mass, and accounted for about 10% of its variation, with nematode populations in roots ranking MC>MF>MK>MI>MS>MA. Useful resistance to ARD and parasitic nematodes apparently exists within Malus germplasm collections, and can be identified by testing more genotypes, developing rapid resistance screening methods, and comparing RLN host preferences among Malus genotypes and various orchard cover crops.

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Stan C. Hokanson, Amy K. Szewc-McFadden, Warren F. Lamboy and James R. McFerson

A diverse collection of 133 Malus species and hybrids from the USDA Plant Genetic Resources Unit's core subset collection was screened with five simple sequence repeat (SSR) primer pairs in order to determine genetic identities and overall levels of genetic variation. The number of amplification products (alleles) per locus (primer pair) in this collection ranged from 6 to 39, with some genotypes showing complex banding patterns of up to four products per locus, suggesting that duplication events may have occurred within the genome. Five primer sets unequivocally differentiated all but 10 pairs of genotypes in the collection, with seven of these 10 being pairs of the same species. Within three of the species holdings surveyed, M. honanensis, M. sargentii, and M. sikkimensis, no genetic variation was revealed with the SSR markers. The discrimination power for the combined loci in this collection was nearly one, which indicates that the likelihood of two genetically different accessions sharing the same alleles at all the loci included in this study would be nearly impossible. Coupled with results from a previous survey of M. × domestica accessions, this finding suggests that with five SSR primer pairs, the majority of the Malus holdings could be assigned a unique fingerprint identity. The average direct count heterozygosity over all loci was 0.620, ranging in value from 0.293 to 0.871 over individual loci. These heterozygosity counts will be compared with a survey of naturally occurring M. sieversii to determine whether current repository holdings are representative of the overall levels of diversity occurring in Malus. Information generated with this study, coupled with passport and horticultural data will inform curatorial decisions regarding deaccessioning of duplicate holdings and plans for future germplasm collections.

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James J. Luby, Peter A. Alspach, Vincent G.M. Bus and Nnadozie C. Oraguzie

Incidence and severity of fire blight [Erwinia amylovora (Burr.) Winslow, Broadhurst, Buchanan, Krumwiede, Rogers, and Smith] following field infection were recorded using families resulting primarily from open-pollination of Malus sylvestris (L.) Mill. var.domestica (Borkh.) Mansf. cultivars and a few other Malus Mill. sp. The families were structured as three sublines, planted in three successive years (1992 to 1994), of a diverse population of apple germplasm established at HortResearch, Hawkes Bay, New Zealand. The incidence of fire blight varied among the sublines with the oldest planting exhibiting more fire blight. Flowering trees were more likely to be infected than nonflowering trees, in terms of both incidence and severity. Furthermore, the level of fire blight was related to flowering date, with later flowering trees having higher levels. Thus, family means and narrow-sense heritability estimates were computed after first adjusting the fire blight score for flowering date by fitting a linear model. Provenance of origin of the maternal parent explained little variation except that M. sieversii Lebed. families were more resistant than M. sylvestris var. domestica families in one subline. Family means computed using all trees, and those from only flowering trees were highly correlated. Families from open-pollination of M. honanensis Rehder and M. xhartwiggii Koehne females were among the more susceptible. Those from several European M. sylvestris var. domestica cultivars as well as from M. baccata (L.) Borkh. and M. toringoides (Rehder) Hughes females were among the more resistant families. Narrow-sense heritability estimates ranged from 0.05 to 0.85 depending on the subline, with most estimates between 0.12 and 0.36. They were higher in the two older sublines that consisted primarily of open-pollinated families from M. sylvestris var. domestica, and lower in the younger subline that consisted primarily of M. sieversii, due to lower incidence and severity in the latter subline. Breeders who consider potential complications of juvenility, tree size, and flowering date in relation to infection periods should be able to exploit field epidemics to perform effective selection.

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Dorcas K. Isutsa and Ian A. Merwin

We tested 40 seedling lots and 17 clonal accessions—representing 941 genotypes and 19 species or interspecific hybrids of Malus—for their resistance or tolerance to apple replant disease (ARD) in a mixture of five New York soils with known replant problems. Total plant biomass, root necrosis, root-infesting fungi, and root-lesion nematode (RLN; Pratylenchus penetrans Cobb) or dagger nematode (DN; Xiphinema americanum Cobb) populations were evaluated in apple seedlings and clones grown for ≈60 days in the composite soil. In addition to phytophagous nematodes, various Pythium, Cylindrocarpon, Fusarium, Rhizoctonia and Phytophthora species were isolated from roots grown in the test soil. Plant growth response was categorized by a relative biomass index (RBI), calculated as total plant dry weight in the pasteurized field soil (PS) minus that in an unpasteurized field soil (FS), divided by PS. Nematode reproduction on each genotype was defined by a relative reproduction index (RRI), calculated as final nematode populations in roots and soil (Pf) minus initial soil populations (Pi), divided by Pi. The RBI, RRI, and other responses of accessions to ARD soil were used to rate their resistance, tolerance, or susceptibility to apple replant disease. None of the accessions was completely resistant to ARD pathogens in our test soil. Seedling accessions of M. sieversii Roem. and M. kirghisorum Ponom. appeared to have some tolerance to ARD, based upon their low RRIs and RBIs. Three clonal rootstock accessions (G.65, CG.6210, and G.30), and four other clones (M. baccata Borkh.—1883.h, M. xanthocarpa Langenf.—Xan, M. spectabilis Borkh.— PI589404, and M. mandshurica Schneid.—364.s) were categorized as tolerant to ARD. The disease response of other accessions was rated as susceptible or too variable to classify. We concluded that sources of genetic tolerance to ARD exist in Malus germplasm collections and could be used in breeding and selecting clonal rootstocks for improved control of orchard replant pathogens.