Evaluation of resistance to verticillium wilt in diploid,wild potato interspecific hybrids

Summary Verticillium wilt (V. albo-atrum Reinke & Berthold or V. dahliae Kleb) threatens potato (Solanum tuberosum L.) production in most growing areas of the world. Genetic resistance offers the most cost-effective and environmentally-sound control measure. However, there is a dearth of genetic and breeding information on resistance to verticillium wilt in potato, because of obscure parentage of some standard cultivars and the complex segregation at the tetraploid level. The wide range of genetic variability in wild relatives of potatoes can be potentially useful as a source of disease resistance, such as verticillium wilt resistance. Six diploid, wild, interspecific Solanum hybrids involving resistant x resistant and susceptible x resistant crosses, were assayed for verticillium wilt resistance under greenhouse conditions to evaluate their potential as sources of verticillium wilt resistance. The cross between S. gourlayi Oka. and S. chacoense Bitt. and its reciprocal had the most resistant progenies based on mean colony counts. No simple mode of inheritance can be proposed based on the observed segregation ratios. However, the continuous distributions observed on verticillium wilt disease response among hybrid families indicate that inheritance of resistance may be polygenic and complex. In addition, skewness of colony count distributions toward the resistance parents were characteristic of all resistant x susceptible crosses suggesting that resistance may be dominant. By contrast, the susceptible x susceptible cross showed a more normal distribution. Overall, the cross between S. gourlayi and S. chacoense showed the most potential as a source of verticillium wilt resistance.     

Transmission of Verticillium wilt resistance to tetraploid potato via unilateral sexual polyploidization

Summary Verticillium wilt is a serious disease of potato and is caused by the soil-borne fungi Verticillium dahliae and V. albo-atrum. No major cultivar is resistant to this disease. Two diploid interspecific potato clones, C287 and C545, were previously identified with consistently high levels of Verticillium wilt resistance and are thought to have the same genotype for the loci that confer resistance to V. dahliae stem colonization. The purpose of this study was to determine whether resistance to V. dahliae stem colonization could be transferred to the tetraploid level in potato via unilateral sexual polyploidization (USP). Progenies in eight families obtained by crossing C287 and C545 to two tetraploid breeding clones, S438 and S440, and the cultivar ‘Atlantic’ were planted in a V. dahliae-infested field and a field without a significant V. dahliae infestation. Resistance was evaluated relative to C545 and C287. There were differences among families for stem colonization and yield. No differences between the mean stem colonization of C545 and C287 progeny were detected. Family differences due to the tetraploid parents indicate that they contributed resistance to the progeny. Stem colonization data from this experiment were consistent with the proposed complementary two-gene model for Verticillium wilt resistance in the diploid parents. Unilateral sexual polyploidization is an effective method for transferring V. dahliae stem colonization resistance to the tetraploid level.     

Application of cell and tissue culture and in vitro selection for disease resistance breeding — a review

Summary Somaclonal variation, i.e. the variation induced by cell and tissue culture, offers an opportunity to broaden the genetic variation of crops. As a result of somaclonal variation a wide range of plant characteristics can be altered. However, the selection of agronomically important traits, e.g. disease resistance, has many limitations. The efficiency of selection can be increased by the application of in vitro selection procedures. Selection strategies that may be applied to obtain disease resistant somaclonal variants are described. Their merits and limitations, in relation to the efficiency of the procedures, the frequency of disease resistant variants and the genetics of the resistance obtained, are discussed.     

Transfer of resistance to Verticillium dahliae Kleb. from Solanum torvum S.W. into potato (Solanum tuberosum L.) by protoplast electrofusion

Summary Interspecific somatic hybrid plants were regenerated after electrofusion of mesophyll protoplasts with the objective of transferring resistance to Verticillium dahliae from Solanum torvum into potato. Early selection of the putative hybrids was based on differences in cultural behaviour of the parental and hybrid calli (particularly the ability of the latter to regenerate early) in combination with morphological markers. Four putative hybrids were recovered from hundreds of calli, probably resulting from complementation of the two parental genomes. The regenerates were tetraploids (2n=4×=48 chromosomes) and exhibited intermediate traits including leaf form, plant morphology and the presence of anthocyanin. The hybrid nature of the four selected plants was confirmed by examining isoenzyme patterns for isocitrate dehydrogenase (Idh), malate dehydrogenase (Mdh), phosphoglucoisomerase (Pgi) and 6-phosphogluconate dehydrogenase (6-Pgd). While the hybrid plants rooted readily and grew vigorously under in vitro conditions, in the greenhouse their development and growth were retarded by difficulties in rooting. When grafted on potato or S. torvum rootstocks, the hybrid plants recovered normal development and growth. Again, they exhibited intermediate morphological traits. Tests for resistance realized in vitro with medium containing 50% Verticillium wilt filtrate showed that all the somatic hybrids were resistant to the fungus filtrate.     

Sources of resistance to Verticillium wilt in Capsicum

Summary A total of 125 novel accessions of Capsicum annuum L. and Capsicum baccatum L. were screened for sources of resistance to Verticillium wilt (Verticillium dahliae Kleb). A soil infestation method with 2000 microsclerotia of V. dahliae per gram of planting medium, and a soil temperature of 25±3°C, identified 27 Capsicum accessions with resistance to Verticillium wilt. P.I. 215699 (a mixture of Capsicum baccatum var. microcarpum and Capsicum annuum), P.I. 535616 (Capsicum annuum), and P.I. 555614 (Capsicum annuum), had the lowest disease severity and the highest percentage of resistant plants.     

Resistance to Pseudomonas solanacearum in the potato: I. Effects of sources of resistance and adaptation

Summary F1 hybrid true potato seed progenies with multiple sources of specific resistance to Pseudomonas solanacearum and adaptation were evaluated under tropical greenhouse conditions for resistance to a race 1 isolate of P. solanacearum. Results indicated that genes for adaptation are involved in conferring resistance to bacterial wilt. The effect of a particular resistant parent clone on the resistance level of its progeny depended on the resistance, adaptation or the combination of both characteristics of its mate. A heat tolerant parent gave a higher frequency of resistant offspring in combination with an ascertained source of resistance. Combining ability was an apparent feature of resistance to bacterial wilt and resistance was highest in the majority of the crosses that had a wide genetic background for both resistance and adaptation. The possible genetic nature of the resistance to bacterial wilt is discussed.     

The potential for early generation selection to identify potato clones with resistance to Verticillium wilt

Verticillium wilt (VW) of potato, caused primarily by the fungus Verticillium dahliae, results in yield loss and is therefore an important soil-borne disease. Resistance to VW exists in potato germplasm and is used by breeders during cultivar development. Breeders could make more rapid progress toward the development of VW resistant clones if they had an effective early generation selection strategy. The purpose of this study was to determine whether selection for VW resistance could be carried out in the first tuber generation on single hills. One hundred and fifty-two clones from 19 families were planted as single hills on a V. dahliae-infested field. Each plant was scored for vine maturity, VW symptom expression, yield, stem colonization (colony forming units (cfu), in dried basal stem segments) and incidence (percent infected stems). In the second clonal generation, which consisted of replicated four-hill plots, stem colonization scores and incidence values were used to identify clones which were more resistant than a moderately resistant cultivar and others which were more susceptible than a susceptible cultivar. The efficiency and reliability of the single-hill selection strategy, based on symptoms and yield, was then determined by comparison to the four-hill results. We determined that the best single-hill selection strategy was negative selection (discard clones with the lowest performance) with low stringency, based on yield.     

Assessment of Gossypium sturtianum and G. australe as potential sources of Fusarium wilt resistance to cotton

When challenged with Fusarium oxysporum f. sp. vasinfectum (Fov) from vegetative compatibility groups (VCGs) 01111 and 01112 in glasshouse tests, Gossypium australe Mueller and Gossypium sturtianum Willis accessions showed a variety of disease responses ranging from highly resistant to highly susceptible. Under high disease pressure G. sturtianum accession Gos-5275 was significantly more resistant than the commercial G. hirsutum cultivars that are designated standards for Fusarium resistance by Australian cotton breeders. Under low disease pressure G. sturtianum accession Gos-5250 was more susceptible than a highly susceptible commercial cultivar. A series of glasshouse tests was performed at two locations (Indooroopilly, QLD. and Canberra, ACT), and under low and high disease pressure. In these tests, a hexaploid cross (Gos-5271) generated from a Fusarium-resistant G. sturtianum (Gos-5275) and a Fusarium-susceptible G. hirsutum L. (CPI-138969) was significantly more resistant to Fusarium wilt than its G. hirsutum parent. Thus G. sturtianum, with a diploid genome and a range of responses to Fov challenge, has the potential to provide the basis for the elucidation of the genetic basis of resistance to Fusarium wilt in cotton species. In addition, resistant accessions of G. sturtianum are identified as a potential source of Fusarium wilt resistance genes for cotton breeding. In the glasshouse tests used to assess the resistance of various Gossypium accessions to Fusarium wilt disease, the scoring of vascular browning was found to give a more reliable indication of disease severity than the scoring of foliar symptoms. This revised version was published online in August 2006 with corrections to the Cover Date.     

In vitro techniques for selecting wheat (Triticum aestivum L.) for Fusarium-resistance. II. Culture filtrate technique and inheritance of Fusarium-resistance in the somaclones

Summary Calli of resistant, intermediary and susceptible wheat (Triticum aestivum L.) varieties were selected using culture filtrates of Fusarium graminearum and F. culmorum and the regenerants were evaluated for resistance up to R₃. Czapek-Dox broth medium was inoculated with mycelia of Fusarium isolates and incubated for 2–6 weeks. Filtrates were added to MS callus growing medium, then 5 weeks-old calli were transferred onto this medium (MST) for 4–5 weeks. MST containing 30% filtrate was found to be suitable for selection. Resistant calli were transferred again to fresh MST for further two selection cycles. The surviving calli produced less fertile regenerated lines (R₀) than the non-selected ones. Among 18 R₁ lines tested for Fusarium-resistance in the seedling stage by artificial inoculation in the greenhouse, two (11.1%) were significantly more resistant, one (5.6%) was more susceptible than the original cultivar and the rest (83.3%) behaved similarly to the donor plants. Among unselected R₃ lines of three varieties, practically the same number of resistant plants were found as among the related selected ones. When the R₃ selfed generations obtained through double-layer and culture filtrate selection techniques were tested for Fusarium-resistance, 35.7% of the lines were found to be more resistant than the original cultivars, none was more susceptible and 64.3% had a reaction similar to that of the source materials. Thus, inheritance of the disease reaction was not stable in all cases. Success of in vitro selection for Fusarium-resistance depended also on the genotype, and toxin analysis showed that although being effective, the selective media contained deoxynivalenol only exceptionally. In selecting wheat for Fusarium-resistance in vitro, the culture filtrate technique proved better than the double-layer procedure.     

Recurrent selection for resistance to Verticillium dahliae in pepper

Summary Two cycles of recurrent selection were performed in a population including 7 small fruited varieties of pepper (Capsicum annuum) with low resistance level to Verticillium and resistance to other soilborne pathogens. The collaboration of 7 plant breeders in this work and the use of 2 artificial methods of inoculation allowed screening of numerous plants for resistance to V. dahliae under different conditions. The population was simultaneously improved for resistance to V. dahliae and for resistance to Phytophthora capsici, and at each cycle, intercrosses were performed between all these plants. The general level of resistance to V. dahliae increased continuously during the 2 cycles of seles of selection. Selfed progenies of plants from the 2 cycles displayed a higher resistance than the original parents, whatever the method of inoculation used. This gave evidence of transgressions for resistance. Crosses involving parents that did not seem resistant by themselves also produced transgressive lines, indicating that resistance genes were present but not active in these varieties. The transgressive lines with polygenic resistance to V. dahliae were included into a further breeding program to introduce genes of agronomic value.     

Interactions between plant and aphid genotypes in resistance of lettuce to Myzus persicae and Macrosiphum euphorbiae

Summary Six lettuce lines, representing two types of resistance to the green peach aphid, Myzus persicae, and a control line with high susceptibility to M. persicae were tested for resistance to six different clones of Myzus persicae and two clones of the potato aphid, Macrosiphum euphorbiae.The clones of M. persicae showed very different levels of aggressiveness on lettuce: two had a high level of reproduction, two had an intermediate level and two were poorly adapted to lettuce as a host. Differences between lettuce lines in aphid reproduction increased with increasing aggressiveness of the aphid clone, which means that aggressive clones are most effective for selection purposes. No evidence was found for clone-specific plant genotype reactions, meaning that lines resistant to one clone will also be resistant to other clones of M. persicae, allthough not neccessarily at the same level. The lettuce lines selected for partial resistance to the aggressive clone WMp1 were completely or almost completely resistant to less aggresive clones.No differences in level of reproduction were found between the two clones of M. euphorbiae and no relation was observed between resistance to M. persicae and M. euphorbiae, indicating the species-specific character of resistance to leaf aphids in lettuce.     

Screening of melon (Cucumis melo L.) germplasm for multiple disease resistance

Summary Melon germplasm was screened for cucumber green mottle mosaic virus (CGMMV), powdery mildew (Sphaerotheca fuliginea), downy mildew (Pseudoperonospora cubensis) and Fusarium wilt (Fusarium oxysporum f. sp. melonis) resistance under artificial conditions except downy mildew for which screening was done under natural epiphytotic conditions. High level resistance to all the four diseases was not recorded in any of the collections tested. Nevertheless, ertheless, resistance to three diseases was located in three germplasm. Wild Cucumis species C. figarei exhibited absolute resistance to CGMMV and Fusarium wilt and high level resistance to downy mildew. Phoot or snapmelon (Cucumis melo var. momordica) — a non-dessert from of Indian origin—was highly resistant to downy mildew and resistant to CGMMV and medium resistant to Fusarium wilt. Iroquois was resistant to powdery mildew and medium resistant to downy mildew and CGMMV.     

Nematode resistance in bananas: screening results on some new Mycosphaerella resistant banana hybrids

Banana hybrids with resistance to Yellow Sigatoka and Black Leaf Streak disease were evaluated for resistance to the burrowing nematode Radopholus similis and to the lesion nematode Pratylenchus coffeae in a growth chamber at 24–28°. Plants produced by tissue culture were acclimatised for 6 weeks prior to inoculation. Forty-five days after inoculation with nematodes, the root systems were processed and nematode numbers assessed. Two cultivars of Grande Naine (Musa AAA, Cavendish subgroup, ITC1256 and cv902) and one cultivar of Yangambi Km5 (Musa AAA, Ibota subgroup, ITC1123) were used respectively as susceptible and resistant controls. Results based on multiplication rates and root infestations showed that three of these hybrids (FB918, FB919 and FB924) were not significantly different from the resistant control Yangambi Km5 with a lower multiplication of R. similis. Similarly four of these hybrids (FB918, FB919, FB920, FB924) showed a lower multiplication of P. coffeae, not significantly different from the same resistant control. This is the first study that shows a partial resistance to both nematode species, R. similis and P. coffeae within synthetic hybrids of M. acuminata, adding an important extra value to these dessert banana hybrids formerly bred to resist to Mycosphaerella leaf spot diseases.     

Inheritance of resistance in soybean PI 567516C to LY1 nematode population infecting cv. Hartwig

Worldwide, cyst nematode (SCN) Heterodera glycines is the most destructive pathogen on cultivated soybean (Glycine max (L.) Merr.). In the USA yield losses in 2001 were estimated to be nearly 60 million dollars. Crop losses are primarily reduced by the use of resistant cultivars. Nematode populations are variable and have adapted to reproduce on resistant cultivars overtime because resistance primarily traces to two soybean accessions. Recently cv. Hartwig was released which has comprehensive resistance to most SCN populations. A virulent nematode population LY1 was recently selected for its reproduction on Hartwig. LY1 population originated from a mass mating of Race 2 (HG Type 1.2.5-) females with Race 5 (HG Type 1.2-) males. LY1 nematode population infects currently known sources of resistance except PI 567516C. The female indices obtained on PI 567516C and Hartwig were 7% (resistant) and 155% (susceptible), respectively. However, the genetic basis of LY1 resistance in soybean PI 567516C is not known. Resistant PI line 567516C was crossed to susceptible cultivar Hartwig to generate 105 F_2:5 families. These families together with parents, seven indicator lines and a susceptible control cv. Lee-74 were evaluated for response to LY1 nematode population in the greenhouse. Chi-square analysis showed resistance in PI567516C to LY1 was conditioned by one dominant and two recessive genes (Rhg, rhg, rhg). Chi-square value was 0.15 and P = 0.70. This information will be useful to soybean researchers for developing resistant cultivars to nematode population that infects Hartwig.     

Expression in vitro of resistance to Heterodera schachtii in hairy roots of an alien monotelosomic addition plant of Beta vulgaris,transformed by Agrobacterium rhizogenes

Summary Hairy roots, induced by Agrobacterium rhizogenes, were obtained of nematode susceptible beet plants (Beta vulgaris) and of the nematode resistant alien monotelosomic addition AN5, carrying a telosome from B. patellaris. The additional telosome was found to be stably present in vitro in the roots of AN5. The hairy root cultures were inoculated with larvae of the beet cyst nematode Heterodera schachtii. On the root culture of AN5 significantly less cysts developed than on the other root cultures. These results indicate that the resistance to the beet cyst nematode is expressed in the roots after transformation and can be monitored under in vitro conditions.     

Evaluation of the virulence of races 1, 2 and 4 of Fusarium oxysporum f.sp. dianthi in carnation

Summary A simultaneous analysis of the virulence of races 1, 2 and 4 of Fusarium oxysporum f.sp. dianthi to a series of nine carnation cultivars revealed the presence of different interactions between races and cultivars, as well as differences in pathogenesis between race 1 on the one hand and race 2 and 4 on the other.The most common race 2 induced typical symptoms of Fusarium wilt in all susceptible cultivars. The cultivars showed considerable variation in resistance to race 2. Only Novada remained free of external symptoms throughout the experiment. In diseased plants of all cultivars studied, infected vascular tissue was white with dark brown margins, and heavy degradation of the cell walls and xylem parenchyma cells had occurred. All Dutch isolates corresponded with race 2.Race 4 induced wilt symptoms similar to those induced by race 2, and there was a similar variation in resistance to race 2 and 4 in the cultivars. On average, the race 4 isolates were less aggressive than those of race 2. Compared with race 2, there was evidence of some genotype × race interactions: Pallas proved to be considerably more susceptible, and Lena more resistant to race 4 than to race 2. The isolates of race 4 induced a nistopathology similar to that induced by race 2, but with less vascular browning.Race 1 induced atypical but severe wilt symptoms and unusual vascular discoloration in Elsy, Niky and Sam's Pride only. The vascular tissue in these cultivars turned pale brown; in spite of heavy colonization of these tissues virtually no degradation of cell walls was observed. All other cultivars tested proved virtually resistant to race 1, providing further evidence for genotype × race interactions.Within races, limited but statistically significant genotype × isolate interactions were found as well, in particular within race 4. These are tentatively attributed to independent variation of two (or more) resistance components.     

A demonstration of the inheritance of field resistance to lettuce downy mildew (Bremia lactucae Regel.) in progeny derived from cv. Grand Rapids

Summary The inheritance of field resistance to downy mildew in lettuce derived from cv. Grand Rapids was studied in F₂, F₃ and F₄ progeny from a cross with the more susceptible cv. Lobjoits Green Cos. The results for 32 randomly produced F₃ families and the F₂ population corresponded closely although the absolute level of resistance varied from season to season according to disease pressure and environmental conditions. The field resistance character showed a quantitative pattern of inheritance but the rapid response to selection at F₃ implied that only a few genes may be involved. F₄ families raised from families selected from the two extremes of the F₃ distribution fell into distinct categories with resistance levels corresponding to that of their F₃ parents. An association between resistance and morphological features of the resistant parent was present at F₂ but not in subsequent generations when individuals and families which were as resistant as cv. Grand Rapids and yet bore little morphological resemblance to this cultivar were readily recovered.     

Assessment of somaclonal variability in hop (Humulus lupulus L.) in vitro meristem cultures and clones by molecular methods

In vitro meristem tissue cultures are used for production of virus-free rootstocks of hop (Humulus lupulus L.). Because use of plant tissue cultures is associated with occurrence of somaclonal variability, we assessed somaclonal variability in hop meristem in vitro cultures before and after thermotherapy by different molecular methods (RFLP, RAPD, STS, ISSR and AFLP) and compared it with existing clonal variability of Osvald's clones 31, 72 and 114. No molecular differences were observed between mother plants and in vitro mericlones by RFLP and STS analyses. Amplified molecular differences were found in RAPD and ISSR products of one from five in vitromericlones cvs. Eroica (E5) and Southern Brewer (SB2), respectively. Similarities with mother plants were 0.965 and 0.913 (JSC), respectively. Specific amplified polymorphic products were found for every mericlone and mother plant in AFLP reactions and variability of DNA sequence ranged from 0.824 to 0.993 (JSC). This variability was very similar to determined intra-clonal variability within Osvald's clones 31, 72 and 114 by AFLP analysis. Inter-clonal variability of DNA sequence was exactly higher than intra-clonal variability of DNA sequence in these clones. The molecular differences between Osvald's clone 72 normal and meristem derived were not verifiable. Thermotherapy increased frequency of molecular changes, since amplified differences were found in 14 from 20 in vitro mericlones of cv. Eroica, in 6 from 11 in vitro mericlones of cv. Yeoman and in 15 from 23 in vitro mericlones of cv. Southern Brewer by RAPD and ISSR analyses.     

Induction of novel organelle DNA variation and transfer of resistance to frost and Verticillium wilt in Solanum tuberosum through somatic hybridization with 1EBN S. commersonii

Somatic hybridization can be used to induce genetic variability in plastidial and mitochondrial genomes, and transfer multiple uncloned genes across sexual barriers. Somatic hybrids were produced between a dihaploid clone of the common potato, S. tuberosum subsp. tuberosum, and the wild sexually incongruent diploid species S. commersonii. Fusion products were selected on the basis of callus growth and regeneration in vitro. Genome composition of putative somatic hybrids was determined by flow cytometric analysis of nuclear DNA content, RAPD analysis, and Southern analysis with probes specific to organellar DNA. All regenerated fusion products proved to be hybrids based on RAPD analysis. Seventy per cent of somatic hybrids were (near) tetraploids, 22% (near) hexaploids and 8%(near) octoploids. A high correlation was found between the nuclear DNA content determined by flow cytometry and the number of chloroplasts in stomata guard cell pairs. Somatic hybrids inherited the parental plastids in a random manner. On the contrary, they preferentially inherited the mitochondrial DNA fragments of S. tuberosum. The majority of them had a rearranged mitochondrial genome with fragments from both parents. Hybrids were highly vigorous and morphologically more similar to the cultivated than to the wild parent, produced tubers on long stolons under long photoperiod conditions, showed a high degree of flowering, but did not produce pollen. In addition, somatic hybrids were generally more resistant to frost and Verticillium wilt than the cultivated parent, indicating the introgression of relevant resistance genes from the wild species into the genetic background of S. tuberosum. This revised version was published online in July 2006 with corrections to the Cover Date.     

Influence of plant age,disease incubation time,and host plant resistance on Verticillium wilt symptom expression in lucerne (Medicago sativa)

Summary The most economical means to control Verticillium wilt in lucerne, caused by Verticillium albo-atrum, is through resistant cultivars. Most Verticillium wilt resistance evaluations use some form of root-cut-soak inoculation procedure. In Trial 1, three lucerne cultivars were inoculated at eight plant ages and plant symptoms were scored at ten successive weekly intervals. Trial 2 consisted of two cultivars inoculated at ten plant ages and symptoms were evaluated at eight weekly intervals. In an experiment to study the genetics of resistance to this pathogen, four plants showing no visual symptoms were selected from each of four germplasms. The selected populations were crossed in diallel fashion. The population crosses plus the parents were evaluated for resistance to V. albo-atrum. Results from Trial 1 and Trial 2 showed disease severity increased with increasing inoculation age and with increasing scoring age. Cultivar symptom expression interacted with inoculation age, but not with scoring age. The diallel analysis supported resistance to Verticillium wilt being conferred by additive gene action, as heterosis was not detected and general combining ability mean square was 3.75 times larger than the specific combining ability mean square.