Fungitoxic effect of scopolin and related coumarins on Sclerotinia sclerotiorum. A way to overcome sunflower head rot

Summary The content of coumarins, as probable phytoalexins, was analysed in four sunflower genotypes that ranged in responses to head rot from highly susceptible to highly resistant. Low levels of all coumarins (scopolin, scopoletin and ayapin) were detected in the three most susceptible genotypes irrespective of time after inoculation. However, in the resistant genotype there was a clear time-dependent disease-induced increase of all coumarins that reached a maximum after 10–14 days. Detailed comparison of the most susceptible and the resistant genotype showed that in the resistant but not the susceptible, scopoletin peroxidase activity increased during the course of the experiment. Results confirmed a clear negative correlation between coumarin content and disease symptoms and in particular for scopolin. Furthermore we show for the first time that scopolin is inhibitory to Sclerotinia at similar doses to scopoletin. As scopolin is known to be less phytotoxic than ayapin and scopoletin, its accumulation may well confer head rot resistance with minimal plant damage and might be one of the bases for resistance to Sclerotinia.     

Effect of common bunt on the frost resistance and winter hardiness of wheat (Triticum aestivum L.) lines containing Bt genes

In order to determine the effects of bunt inoculation on frost resistance and winter hardiness in lines containing resistance genes, the bunt [Tilletia foetida (Wallroth) Liro, T. caries (DC.) Tulasne] susceptibility of wheat lines containing bunt resistance genesBt1 to Bt10 and the effect of the year on the degree of infection were studied over six years from 1991 to 1997 in an artificial inoculation nursery. Uninoculated and artificially inoculated wheat plants were tested for frost resistance in the phytotron in 1995 and in the field in boxes in three years from 1994/95 to 1996/97. The line withBt10 was very resistant, lines with Bt5, Bt6, Bt8 and Bt9 were resistant, the line with Bt4 was moderately resistant, those with Bt2 and Bt3 were moderately susceptible, the line with Bt1 was susceptible and the line with Bt7 was very susceptible to the local bunt population in Hungary. Bunt incidence also varied over years. The frost resistance of the Bt lines was generally lower after bunt inoculation than that of uninoculated plants. The increased frost kill in inoculated plants was not correlated with the extent of varietal susceptibility to bunt. Some lines with resistance, namely those with Bt5 (1.6% infection), Bt8 (0.6%) and Bt10 (0.0%), suffered significantly greater frost kill in the young plant stage as the result of bunt inoculation. By contrast, the Bt7line had excellent frost resistance and winter hardiness but suffered the greatest extent of bunt infection, whereas the Bt6 line had good frost resistance and good bunt resistance. This revised version was published online in July 2006 with corrections to the Cover Date.     

Colocation of downy mildew (Plasmopara halstedii) resistance genes in sunflower (Helianthus annuus L.)

Summary The Pl6 locus in the inbred sunflower (Helianthus annuus L.) line HA335 giving resistance to French races of downy mildew (Plasmopara halstedii (Farl.) Berl. & de Toni. was localized by molecular techniques. A bulked segregant analysis was made on the F2 progeny from a cross between this line and H52, a downy mildew susceptible line. The resistance gene in HA335 was found to have the same linked RFLP marker loci as those determined for Pl1 (resistance to race 1 in the line RHA266) on linkage group 1 of the consensus RFLP map of the cultivated sunflower. Pl1 and Pl6 thus appear either to be allelic or closely linked. The implications for sunflower breeding are discussed.     

Inheritance of resistance to Botrytis cinerea Pers. in Cicer arietinum L.

Summary Chickpea (Cicer arietinum L.) line ICC 1069 was selected as resistant parent after screening for resistance to grey mould (Botrytis cinerea Pers.) under artificial inoculation conditions. It was crossed with four high yielding susceptible varieties of chickpea. Crosses ICC 1069 × BGM 413 and ICC 1069 × BG 256 showed monogenic dominant resistance in ratio of 3R (resistant): 1S (susceptible). However, in crosses, ICC 1069 × BGM 419 and ICC 1069 × BGM 408, a ratio of 13S (susceptible) : 3R (resistant) was obtained indicating the presence of epistatic interaction. The results pointed towards the presence of a type of major gene resistance to grey mould in chickpea.     

Inheritance of resistance to two races of sunflower downy mildew (Plasmopara halstedii) in two Helianthus annuus L. lines

Sunflower downy mildew caused by Plasmopara halstedii is an important disease of sunflower capable of causing losses of more than 80% of production. Races 100, 300, 310, 330, 710, 703, 730 and770 of the fungus have been identified in Spain. Race 703, of high virulence, has been identified frequently in the northeast, while race 310 seems to occur over the south, the main sunflower growing region of the country. Oil sunflower lines RHA-274 and DM4 were studied for their resistance to races 310(RHA-274 and DM4) and 703 (DM4). In each cross, only one plant of the resistant parent was crossed to the inbred susceptible line HA-89 (or cmsHA-89).Plants from F₂ and backcross(BC₁F₁ to susceptible parent)generations were evaluated for fungal sporulation on true leaves and/or cotyledons. The resistant-to-susceptible ratios obtained in the F₂ and BC₁F₁ progenies from the crosses cmsHA-89 × RHA-274 and HA-89 × DM4suggested that one major gene in each line is responsible for resistance to race 703.The segregations of the progenies of the cross HA-89 × DM4 inoculated with race 703also fitted the ratios 1:1 and 3:1 (for BC₁F₁ and F₂, respectively)corresponding to control of resistance by a single dominant gene. In RHA-274, the gene for resistance to race 310 was designated Pl ₉, whereas Pl _v is tentatively proposed to designate the gene in DM4 responsible for resistance to races310 and 703. This revised version was published online in August 2006 with corrections to the Cover Date.     

Evaluation of screening techniques and determination of criteria for assessing resistance to Corynebacterium michiganense in tomato

Summary When screening populations segregating for resistance to Corynebacterium michiganense a rapid and reliable inoculation technique is needed. From the several techniques tested, inoculation of the petiole was found to give better differentiation of resistant and susceptible plants than did inoculation of the root or leaf. When petiole-inoculation was compared with stem-inoculation the latter showed the lowest environmental variation and is suggested for use in genetic studies. Stem-inoculation involved clipping the stems of 4 week old seedlings 1 cm above the cotyledonary leaves with scissors which had been dipped in inoculum.Petiole inoculation and, to a lesser degree, stem inoculation failed to produce uniform infection, resulting in variation in the expression of the disease. The scoring was therefore based on the presence, and not the degree, of wilting and stunting, or the size of the cankers. Plants free of all these symptoms were rated resistant. The coefficient of variation for vascular discoloration on individual plants was calculated to be 50%, which in part may be explained by the histopathology of the host.The virulent isolates cm 21 caused death of seedlings resistant to isolate H. However, cultivars resistant to isolate H in the seedling stage did not decrease in fruit weight as compared to susceptible cultivars when inoculated with isolate cm 21 at anthesis.Michigan Agricultural Experiment Station Journal No. 7424.     

A single dominant gene for downy mildew resistance in broccoli

Downy mildew, incited by Peronospora parasitica (Pers.: Fr.) Fr., is a destructive disease of broccoli (Brassica oleraceaL., Italica Group). Resistant cultivars represent a desirable control method to provide a practical, environmentally benign, and long-term means of limiting damage from this disease. Doubled-haploid (DH) lines developed by us exhibit a high level of downy mildew resistance at the cotyledon stage. To determine the mode of inheritance for this resistance, a resistant DH line was crossed to a susceptible DH line to make an F₁, from which F₂ and backcross (BC) populations were developed. All populations were evaluated for response to artificial inoculation with P. parasitica at the cotyledon stage. All F₁ plants (including reciprocals) were as resistant as the resistant parent, indicating no maternal effect for this trait. F₂ populations segregated approximately 3resistant to 1 susceptible, BC populations using the resistant parent as the recurrent parent contained all resistant plants, and the BC to the susceptible parent segregated 1 resistant to 1 susceptible. These results indicate that resistance is controlled by a single dominant gene. This gene should be easily incorporated into F₁ hybrids and used commercially to prevent downy mildew at the cotyledon stage. This revised version was published online in August 2006 with corrections to the Cover Date.     

Resistance of sunflower inbred lines to various forms of attack by Sclerotinia sclerotiorum and relations with some morphological characters

Summary Twenty sunflower inbred lines were studied for their reactions to 7 Sclerotinia sclerotiorum tests on different plant parts. A principal component analysis (P.C.A.) and t-tests on the means of resistant and susceptible groups indicated that reactions of inbred lines to infection of capitula by ascospores are independent from those to ascospore infections of terminal buds. They are even more contrasted with the results of any test measuring mycelial extension. However, for the last, there is a close association between the reactions of roots, leaves and capitula. Of the inbred lines, some showed good levels of resistance to most forms of attack, others were generally susceptible and there were some with good resistance to one particular form of attack. A P.C.A. of 18 morphological and field characters showed no general association between these characters and Sclerotinia sclerotiorum test results, although it was found that lines resistant to mycelial extension on capitula were generally earlier than those that were more susceptible. It is proposed that breeding programmes for general resistance to Sclerotinia sclerotiorum should include a combination of two or three tests.     

Brassica coenospecies: a rich reservoir for genetic resistance to leaf spot caused by Alternaria brassicae

Development of leaf spot resistant mustard cultivars is a relevant objective in view of heavy crop losses caused by this pathogen. Thirty-eight species belonging to 9 genera, including cultivated and wild allies, of the genus Brassica were evaluated under epiphytotic conditions for two years. Inoculations were done on whole plants (in vivo) and on detached leaves (in vitro). Data on incubation period, number of lesions per leaf, lesion size and leaf area covered by lesions were recorded. Species which never produced disease symptoms throughout the growing period in pots and until 72 hours after inoculation in detached leaf assays during both years were treated as resistant, while those that produced symptoms were classified as moderately resistant, susceptible or highly susceptible depending upon incubation period, size of lesions and leaf area covered by disease symptoms. Eight species (Brassica desnottesii, Camelina sativa, Coincya pseuderucastrum, Diplotaxis berthautii, D. catholica, D. cretacea, D. erucoides, and Erucastrum gallicum) were found completely resistant, whereas others were classified as moderately resistant (12), susceptible (11) or highly susceptible (9). Since resistance is unavailable within the cultivated species, these 8 resistant wild species could be used as donor parents for introgressing resistance to leaf spot disease in Indian mustard. This revised version was published online in August 2006 with corrections to the Cover Date.     

Genetical analysis of resistance to Mycosphaerella pinodes in pea seedlings

Summary In studies of the inheritance of resistance, pea seedlings of seven lines in which stems and leaves were both resistant to Mycosphaerella pinodes were crossed with a line in which they were both susceptible. With seven of the crosses resistance was dominant to susceptibility. When F₂ progenies of five crosses were inoculated on either stems or leaves independently, phenotypes segregated in a ratio of 3 resistant: 1 susceptible indicating that a single dominant gene controlled resistance. F₂ progenies of one other cross gave ratios with a better fit to 9 resistant: 7 susceptible indicating that two co-dominant genes controlled resistance. The F₂ progeny of another cross segregated in complex ratios indicating multigene resistance.When resistant lines JI 97 and JI 1089 were crossed with a susceptible line and leaves and stems of each F₂ plant were inoculated, resistance phenotypes segregated independently demonstrating that leaf and stem resistance were controlled by different genes. In two experiments where the F₂ progeny of the cross JI 97×JI 1089 were tested for stem and leaf resistance separately, both characters segregated in a ratio of 15 resistant:1 susceptible indicating that these two resistant lines contain two non-allelic genes for stem resistance (designated Rmp1 and Rmp2) and two for leaf resistance (designated Rmp3 and Rmp4). Evidence that the gene for leaf resistance in JI 1089 is located in linkage group 4 of Pisum sativum is presented.     

Monogenic resistance in tomato to Fusarium oxysporum f. sp. lycopersici race 3

Summary Resistance to fusarium wilt, incited by Fusarium oxysporum (Schlecht.) f. sp. lycopersici (Sacc.) Snyder & Hansen race 3 in tomato (Lycopersicon esculentum Mill.) was discovered in LA 716, a L. pennellii accession. A resistant BC₁F₃ breeding line, E427, was developed from LA 716. E427 was crossed with the susceptible cv. Suncoast and F₁, BCP₁, BCP₂ (to Fla 7155, a susceptible parent) F₂, F₃, and BCP₂S₁ seeds were obtained. Segregation for resistance following root dip inoculation over three experiments indicated a single dominant gene controlled resistance. Five of the 12 BCP₁S₁'s segregated more susceptible plants, whereas one of the 12 segregated more resistant plants than expected (P<0.05). Three of 23 F₃ lines segregated more susceptible plants than expected while 1 of the 23 had more resistant plants than expected (P<0.05). Segregation in all other lines fit expected ratios. Five of the 23 F₃'s were homozygous resistant which was an acceptable fit to expectations (P=0.1–0.5). The gene symbol I ₃ is proposed for resistance to race 3 of the wilt pathogen. Deviations from expected ratios in data reported here and for other breeding lines indicate an effect of modifier genes and/or incomplete penetrance. Plant age at inoculation and seed dormancy did not affect results.Florida Agricultural Experiment Station Journal Series No. 8101.     

Involvement of polyamines, diamine oxidase and polyamine oxidase in resistance of barley to Blumeria graminis f. sp. hordei

Polyamine levels and diamine and polyamine oxidase activities have been investigated in the first leaves of barley (Hordeum vulgare L.) in the absence of or following inoculation with conidia of powdery mildew (Blumeria graminis f. sp. hordei). Two cultivars with varying sensitivity to powdery mildew, viz., Chariot (resistant) and Golden Promise (susceptible) were used. The levels of putrescine, spermidine and spermine were found to be higher in the leaves of Chariot than in the leaves of Golden Promise and, with the exception of spermine, were generally higher in both cultivars after inoculation. In inoculated leaves of Chariot, levels of putrescine and spermidine peaked at 9 days and 12 days, respectively. In controls (uninoculated leaves), the activities of these enzymes, and putrescine and spermidine levels also increased but not to the same extent as in inoculated leaves. With Golden Promise, the levels of putrescine and spermidine in the inoculated leaves changed very little over sampling times but were always higher than in the controls. In this cultivar, there was little difference between inoculated leaves and the controls in diamine oxidase activity which reached a maximum value at 9 days post-inoculation. Activity of the bound form of diamine oxidase was low in both the cultivars. Polyamine oxidase was not detected at 3 days after inoculation in either cultivar but activity at fairly low levels was recorded at later times, usually reaching a maximum value at 9 days. The results suggest that polyamine metabolism and diamine oxidase activity in particular may be involved in the mechanism conferring resistance to barley powdery mildew in Chariot.     

Difference between USA and French isolates of tobacco etch virus and pepper mottle virus displayed by doubled haploid line analysis

Summary Doubled haploid peppers resistant in France to specific strains of tobacco etch virus (TEV) and pepper mottle virus (PeMV) were indexed for resistance to USA strains of TEV and PeMV. The doubled haploids were inoculated when four to six true leaves had developed. The peppers were indexed after an incubation period, and a disease index was calculated. Plants without viral symptoms were reinoculated. Plants challenged to TEV were inoculated four times. Doubled haploids, TEV-resistant in France, were susceptible to the USA strain by the fourth screening. All doubled haploid lines showed TEV symptoms after the fourth inoculation. Two doubled haploids lines, no. 4 and no. 8, had about 6% resistance to TEV after the fourth inoculation. Doubled haploid line no. 5 was 100% resistant to PeMV. The results confirm that the USA PeMV and the USA TEV isolates are different pathotypes than the French ones used originally to screen the doubled haploids. In addition, the use of doubled haploids to identify pathotypes of PeMV and TEV is demonstrated.     

Segregation distortion of Brassica carinata derived black rot resistance in Brassica oleracea

Three segregating F₂ populations were developed by self-pollinating 3 black rot resistant F₁ plants, derived from across between black rot resistant parent line 11B-1-12 and the susceptible cauliflower cultivar ‘Snow Ball’. Plants were wound inoculated using 4 isolates ofXanthomonas campestris pv. campestris (Xcc) race 4, and disease severity ratings of F₂ plants from the three populations were scored. A total of 860 arbitrary oligonucleotide primers were used to amplify DNA from black rot resistant and susceptible F₂ plants and bulks. Eight RAPD markers amplified fragments associated with completely disease free plants following black rot inoculation,which segregated in frequencies far lower than expected. Segregation of markers with black rot resistance indicates that a single, dominant major gene controls black rot resistance in these plants. Stability of this black rot resistance gene in populations derived from 11B-1-12 may complicate introgression into B. oleracea genotypes for hybrid production. This revised version was published online in July 2006 with corrections to the Cover Date.     

Resistance to Sclerotinia sclerotiorum of 'high oleic' sunflower inbred lines

Cultivation of sunflower (Helianthus annuus L.) is strongly affected by Sclerotinia sclerotiorum. To identify new sources of genetic diversity for sunflower breeding 25 sunflower inbred lines were analysed using eight Amplified Fragment Length Polymorphism (AFLP) primer combinations and their genetic similarities (GS) were estimated. Data were used to develop a Unweighted Pair Group Method using Arithmetic Averages (UPGMA) dendrogram. GS values of 0.58‐0.98 were observed but with no separate groupings dependant on oil quality. The inbred lines were screened for their reaction to inoculation with Sclerotinia sclerotiorum (Lib.) de Bary. Sunflower heads were artificially inoculated with S. sclerotiorum in three environments. Head infection was monitored after 1 week (lesion length) and 2 weeks (head rot). The F5 generation of a cross between a resistant (SWS‐B‐04) and a susceptible inbred line (SWS‐B‐01) was also tested for sclerotinia reaction across three environments. Significant differences in sclerotinia resistance, moderate heritabilities and a high correlation between the two assessments were observed. Inbred lines with a high level of resistance could be identified. These lines can be used for further breeding to improve sunflower sclerotinia resistance and to develop superior new hybrids.     

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.     

Identification of sources of resistance in Lycopersicon species to Tomato leaf curl geminivirus (ToLCV) by agroinoculation

A total of 90 genotypes of Lycopersicon species were tested forresistance to the Tomato leaf curlgeminivirus (ToLCV) by agroinoculation andthe vector whitefly (Bemisia tabaciGenn.) inoculation techniques underinsect-proof glasshouse conditions. Therate of infection in the inoculated plantswas determined by detection of the viralDNA in individual plants by the nucleicacid spot hybridization (NASH). Of the 38cultivars and 11 breeding lines of L.esculentum Mill. tested, none was highlyresistant or resistant while three andseven were moderately resistant whenexposed to the cloned virus DNAs byagroinoculation and whitefly inoculationrespectively. On the other hand, among the38 commercial cultivars screened, 16(42.1%) were highly susceptible in vectorinoculations and 31 (81.6%) inagroinoculation. Among the exoticcollection (EC) accessions six were highlyresistant, eleven resistant to whiteflyinoculation and none was highly susceptiblein either of the two tests, indicating thepresence of resistance among the ECaccessions. A higher degree of resistancewas observed in other species of Lycopersicon. While only one accession ofL. cheesmanii Riley was tested, itcould not be infected by either of the twomethods. L. pimpinellifolium (Jusl.)Mill. genotype EC 251580 was similarlyresistant. In L. peruvianum (L.)Mill., five EC accessions could not beinfected by whitefly inoculation, withthree of these being resistant and twomoderately resistant in agroinoculation.This study demonstrates the importance ofthe agroinoculation technique in the virusresistance screening programs andidentifies several good sources ofresistance to the Tomato leaf curlvirus in Lycopersicon species.     

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.     

Expression of resistance to downy mildew at cotyledon and adult plant stages in Brassica oleracea L.

This research compared the expression of resistance to downy mildew at cotyledon and adult plant stages in seven Brassica oleracea genotypes against two P. parasitica isolates of different virulence. Seven day old seedlings were dual inoculated under controlled environment by depositing two 10 μl droplets of a spore suspension of a different isolate on each cotyledon and the interaction phenotype (IP) evaluated 7 days later using a 0–5 scale of increasing susceptibility. The seedlings were transplanted to 16 cm pots and grown in the greenhouse for 110 days (15 to 28 leaves). Adult plants were tested using a single leaf inoculation method that allowed the same plant to be simultaneously inoculated with the two P. parasitica isolates. Leaves were scored 10 days after inoculation using a 0–5 scale of increasing susceptibility. The inoculation of the same plant with the two isolates produced different combinations of cotyledon (CT) and adult-plant (AP) interaction-phenotype according to the genotype. CrGC 3.1 was susceptible at CT and AP stages, ‘Algarvia’ resistant at CT and AP stages, and broccoli ‘A’ susceptible at CT and resistant at AP stage against the two isolates. ‘Murciana’ and broccoli ‘B’ were differential at CT and AP stages. Savoy and Shetland cabbages were differential at CT stage and resistant to both isolates as adult plants. Cotyledon resistance could not be used to predict adult-plant resistance since the two types of resistance were very poorly correlated. Resistance can be race specific either at cotyledon or at adult-plant stage. This revised version was published online in July 2006 with corrections to the Cover Date.     

Inheritance of adult-plant resistance to Phytophthora capsici in pepper

Summary Inheritance studies were conducted to determine the genetic basis of adult-plant resistance in pepper (Capsicum annuum L.) to Phytophthora capsici. F₁, backcrosses and F₂ populations were developed using the resistant parent Criollo de Morellos 334 and susceptible parents Agronômico 10-G and Yolo Wonder. Pepper plants, at 36 days post-emergence, were inoculated near the base of the stem with an inoculum suspension of 5×10⁴ zoospores/ml. Segregation ratios in the F₂ generation of 13 resistant to 3 susceptible plants fit a 2-gene model for resistance with dominant and recessive epistasis.