Abstract
The bacterium Xanthomonas fragariae causes the economically important angular leafspot disease to which a high level of resistance has not been found within the cultivated strawberry Fragaria × ananassa. The 2002 introduction of resistance donors ‘US 4808’ and ‘US 4809’ by the Fruit Laboratory at the Beltsville Agricultural Research Centre provided sources of resistance at the octoploid level derived from F. virginiana. Moving this resistance through three generations of back crossing to different recurrent F. × ananassa parents along with screening progeny for resistance with a spray inoculation method, followed by selection based on horticultural traits, has created improved resistant genotypes. Resistant × susceptible crosses have produced from zero to 21% resistant progeny with a mean of 4.8% for 25 crosses. The presence of undesirable traits, such as small and soft fruit, variegation, pistillate flowers, and susceptibility to powdery mildew, has slowed progress, but this linkage drag can be overcome by increasing screened population size and additional backcross generations. Two resistant × resistant crosses were made in 2009, yielding 32.2 and 32.7% resistant seedlings.
INTRODUCTION
Angular leafspot disease of strawberry (Fragaria × ananassa Duchesne ex Rozier), caused by Xanthomonas fragariae Kenn. & King, is an economically important disease in many production areas worldwide, and effective chemical control measures are unavailable (CitationMaas et al., 1995). The pathogen can be readily spread by nursery stock, often without visible symptoms, so it is a great concern to the strawberry nursery industry. To prevent the introduction of X. fragariae (Xf) to new countries, early detection is crucial. Very sensitive molecular techniques have been developed for this purpose (CitationVandroemme et al., 2008; CitationWeller et al., 2007) and heat treatment has been suggested as a means to eradicate the bacteria from nursery stock (CitationTurechek and Peres, 2009). A high level of plant host resistance to angular leafspot disease has been found in certain Fragaria species, such as the F. moschata Duch. (2n = 6x) and F. vesca L. (2n = 2x) and occasionally in clones of F. virginiana Duch. (2n = 8x) but not in octoploid cultivated strawberries (CitationHildebrand et al., 2005; CitationMaas et al., 1995; CitationXue et al., 2005). Since breeding progress is expected to be more rapid with an octoploid source of resistance, CitationMaas et al. (2002) introduced strawberry clones ‘US 4808’ and ‘US 4809’ as highly resistant parent clones derived from F. virginiana. The inheritance of this resistance was examined in crosses with the highly susceptible cultivar Sweet Charlie after inoculation with four strains of Xf (CitationLewers et al., 2003). Resistance was determined to be recessive to susceptibility and three or four unlinked loci could explain inheritance patterns. The authors noted that “large populations will be needed to identify enough resistant progeny from which to select for other important traits.” We have attempted to use ‘US 4808’ and ‘US 4809’, which both produce small soft fruit, as a starting point to breed commercially acceptable strawberry cultivars.
MATERIALS AND METHODS
Seedling Production and Inoculation
Plants were grown from seed in a soil mix of 107 L Pro-Mix BX to 10 kg perlite under greenhouse conditions of 21°C and natural light supplemented with sodium vapor lamps to give a 14-hr photoperiod. Seed was germinated in 24 × 14 × 7 cm fiber trays and grown for 5 to 6 weeks, then pricked from the soil and transplanted to wooden flats (52 × 30 × 6.5 cm) in six rows spaced 9.5 cm with ten plants spaced 2.8 cm in each row. The seedlings were grown for another 6 weeks until the plants had four to five trifoliate leaves. The seedlings were then transferred to a humid chamber (122 × 213 × 46 cm) constructed of wood framing and covered with clear 4 mil (102 μm) polyethylene located in a growth chamber at a 20:10°C (light:dark) temperature regime and a 14-hr photoperiod of 300 μmol·m−2·s−1. High humidity was maintained by periodically adding water to a capillary mat on the chamber floor. The seedlings were acclimatized to the chamber for 2–3 days prior to inoculation and then inoculated during the morning hours after plants had been exposed to light for a minimum of 4 hr, to ensure that stomata were open and receptive to inoculum (CitationHildebrand et al., 2005; CitationAllan-Woytas et al., 2010). Inoculum was prepared from a local isolate of Xf (designated W2) grown for 5 days on potato dextrose agar and then suspended in water to create a suspension of 108 colony forming units per mL (A 600 = 0.1). The abaxial surfaces of seedling leaves were then thoroughly sprayed with the suspension and plants were returned to the humid chamber. After 3 and 4 weeks, disease severity was estimated on a scale slightly modified from CitationHildebrand et al. (2005) where 0 = no lesions, 1 = 0.33, 2 = 0.67, 3 = 1.3, 4 = 2.7, 5 = 5.3, 6 = 11, 7 = 21, and 8 = 43 lesions cm−2. Seedlings with all leaflets having a rating of less than 2 were transplanted outdoors into field plots.
Crosses
Plants of ‘US 4808’ (a wild F. virginiana from Minnesota) and ‘US 4809’ (derived from a cross between F. virginiana ‘SG26’ and F. × ananassa ‘Earliglow’) were received from Dr. CitationJohn Maas, USDA Beltsville, in 2002. During winter 2003, both resistant parents were pollinated in the greenhouse with pollen of ‘Rosie’, a cultivar with large, symmetrical fruit. Seedlings were grown with a portion being planted in the field and a portion being inoculated with Xf. The five resistant seedlings identified in 2004 in this first round of breeding, either first filial (F1) or first back cross generation (BC1), were back crossed to F. × ananassa parents not closely related to ‘Rosie’. These seedlings, either BC1 or BC2, were screened for resistance and the resistant population planted in the field yielded five selections in 2006. The parents included ‘Tristar’ because among 22 cultivars it had the highest resistance to Xf (CitationHildebrand et al., 2005), and ‘K99-22’ from the Agriculture and Agri-Food Canada breeding program known to contribute large firm fruit to its progeny. A third round of breeding created additional BC2 and BC3 seedlings from which selections were chosen in 2008 and 2010 (). In addition, two advanced generation (resistant × resistant) crosses (designated C4) were made with seedlings screened and resistant genotypes planted out (). Populations from five of the crosses were planted out without screening in 2009, to compare selections from screened and unscreened cohorts.
TABLE 1 Summary of Breeding and Screening for Resistance to X. fragariae
Inoculation of Potted Plants
Runners of the 2010 selections (mostly unscreened as seedlings) were grown in 12.7 cm (5″) pots from runner plants rooted in August and grown in the greenhouse (14-hr photoperiod). Three trifoliate leaves per plant were inoculated as described for seedlings. On each of 5 days in November a different single plant (to give five replicates) from each selection was inoculated, then each leaflet was assessed for disease severity after 3 and 5 weeks incubation.
RESULTS
Three Xf-resistant seedlings of ‘US 4808’ × ‘Rosie’ were identified from 68 seedlings screened in a controlled environment. Similarly, two Xf-resistant seedlings of ‘US 4809’ × ‘Rosie’ were identified from 52 seedlings. These five selections, designated ‘W-1’ to ‘W-5’, are the first filial (F1) (‘W-1’ to ‘W-3’) and first back cross generation (BC1) (‘W-4’ and ‘W-5’) from F. virginiana via the Xf-resistant USDA germplasm ‘US 4808’ and ‘US 4809’, respectively (). An additional 50 seedlings each of ‘US 4808’ × ‘Rosie’ and ‘US 4809’ × ‘Rosie’ were fruited in the field but no selections were taken because of the severity of powdery mildew [caused by Podosphaera aphanis (Wallr.)] on the leaves and the fruit of each plant, indicating high susceptibility.
TABLE 2 Flower Type and Presence of Leaf Variegation of the Resistant Selections Chosen from Seedling Populations Screened for Resistance to X. fragariae
The mean proportion of resistant seedlings in resistant × susceptible crosses of the F1, BC1, BC2, and BC3 generations were 5.5, 8.6, 3.2 and 4.7%, respectively (). Four BC2 and one BC3 crosses gave zero resistant seedlings. The highest proportion resistant was 21.1% in BC1 cross ‘W-2’ × ‘K99–22’ and 18.3% in BC3 cross ‘K06-12’ × ‘K03-2.’ The two resistant × resistant crosses (designated C4 generation) gave 32.2 and 32.7% resistant seedlings.
Twelve 2010 selections from the previously unscreened 252 BC2 and 168 C4 seedlings, following Xf inoculation in pots of rooted runners, gave six resistant genotypes (). Disease severity ratings on these selections were well separated at either end of the severity scale. Those with low disease ratings displayed a few typical lesions 3 weeks after inoculation and occasionally also showed a few atypical lesions that were light tan in color after 5 weeks. However, bacteria did not ooze from these lesions (even after 7 weeks) but when excised, surface sterilized, and plated onto potato dextrose agar, pure cultures of Xf were recovered, indicating the pathogen was able to become established in the tissues at low levels, but not to the extent to cause typical symptoms.
TABLE 3 Flower Type, Presence of Leaf Variegation, and Reaction to Xf Runner Plants Propagated from Selections of BC2 or C4 Crosses not Selected for Xf in the Seedling Stage
All of these 2010 selections had perfect flowers and did not show symptoms of variegation during 2010. Several of these BC2 crosses produced seedlings with large fruit, especially ‘K06-9’ × ‘K06-2’. The largest fruited of these was ‘K10-16’, which had a mean primary fruit weight of 43 g (n = 4); it, however, was susceptible to Xf but its existence suggests that larger populations may contain a resistant genotype with large fruit. Of the 2010 Xf-resistant selections, only ‘K10-15’ was highly susceptible to powdery mildew [caused by Podosphaera aphanis (Wallr.)].
DISCUSSION
Our starting point for developing strawberry cultivars resistant to angular leafspot disease was the Xf-resistant, but horticulturally unimproved, F. virginiana, ‘US 4808’ and F. virginiana × ‘Earliglow’ selection ‘US 4809’, released by the USDA Fruit Laboratory in Beltsville (CitationMaas et al., 2002). ‘US 4808’ and ‘US 4809’ produce small fruit (∼1 to 2 g) with a soft texture, flowers that are pistillate, plants that produce many fine runners, and leaves that are susceptible to powdery mildew. Therefore, in addition to our primary selection criteria—Xf resistance and large, firm fruit—acquiring perfect flowers and avoiding powdery mildew susceptibility were important criteria. Another negative trait that was observed in the BC2 generation from ‘US 4808’ and BC3 from ‘US 4809’ was white streak (variegation). When introgressing wild germplasm into F. × ananassa, the possibility of incorporating unfavorable alleles through linkage drag has been pointed out by CitationStegmeir et al. (2010). CitationBringhurst (1983) emphasized the importance of keeping essential traits at the forefront of selection, and avoiding the dilatory effect of adding non-essential traits. Previous studies have indicated that at least three rounds of crossing back to F. × ananassa were needed before cultivar-quality genotypes were identified (CitationScott and Lawrence, 1975).
Crosses between pistillate genotypes and perfect flowered pollen sources are expected to yield a 1:1 ratio of pistillate to perfect flowered seedlings (CitationAhmadi and Bringhurst, 1991; CitationValleau, 1923). Among seven pistillate × perfect crosses, we recorded 58.5% pistillate seedlings among resistant progeny. Among seven perfect × perfect crosses, in which one grandparent was pistillate, we recorded 13.9% pistillate seedlings among resistant progeny. If only perfect flowered seedlings were selected, the population size would be greatly reduced, so pistillate seedlings were not excluded, especially in the F1 and BC1. Most of the BC2 and BC3 have perfect flowers although, at the time of selection, when fruit is ripe, it is not always apparent whether the selection produces perfect flowers.
In 2007, 1,080 seedlings in four families of the BC2 and five families of the BC3 generation were screened, which identified just 30 resistant seedlings to plant in the field. Of the nine crosses, one had 22 resistant seedlings, another had 4, four crosses each had 1, and three crosses had zero resistant seedlings. By the time of selection from these 30 plants in early July 2008, 11 showed leaf variegation. This low population size of seedlings greatly inhibits progress in selecting genotypes with improved horticultural traits. Consequently, in 2009, in addition to the screened seedlings of five crosses, 84 plants each of the same of five crosses were planted out unscreened. These unscreened seedlings yielded 12 selections, some of which had fruit size and firmness comparable to commercially grown cultivars. Six of these selections were identified as resistant to Xf, all 12 have perfect flowers and as yet, none are showing variegation. This high proportion of resistant selections from unscreened populations was unexpected. For example, 238 seedlings of ‘K06-9’ × ‘K06-2’ were screened but zero resistant seedlings were identified. From the 84 unscreened plants of the same cross, four plants were selected and two of these were subsequently shown to be resistant. Considering all five crosses, only 14% of the 1,312 screened seedlings were resistant—much lower than the 50% resistant of unscreened selections (). One explanation for this may be that some 12-week-old seedlings do not yet express the resistance of adult plants. Adult-plant resistance is present in other pathosystems, for example bacterial blight in rice caused by X. campestris pv. oryzae (CitationQi and Mew, 1985). Further research will determine if adult-plant resistance to Xf occurs in strawberry.
CONCLUSION
With three generations of back-crossing, genes conferring resistance to angular leafspot disease have been introgressed into F. × ananassa. Further testing of the 2010 selections in field plots will determine whether genotypes worthy of cultivar status are present, or whether additional rounds of crossing are required. The high proportion of resistant selections among runners derived from unscreened seedlings of resistant × susceptible and resistant × resistant crosses suggests that screening young seedlings may be too severe and so may discard plants that would be resistant when older. This implies that growing larger populations of unscreened seedlings followed by testing selections for resistance may enhance breeding progress.
ACKNOWLEDGMENT
We thank John Maas for providing plants of ‘US 4808’ and ‘US 4809’, and Michael Graves, Pansy Rand, and Robert Davies for technical assistance in the field and greenhouse. Atlantic Food and Horticulture Research Centre Contribution No. 2386.
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