Use of culture-derived Fusarium oxysporum f. sp. cubense, race 1 filtrates for rapid and non-destructive in vitro differentiation between resistant and susceptible clones of field-grown banana

Banana and plantain are among the most important food crops in developing countries but production is threatened by increasing virulent forms of Fusarium oxysporum f. sp. cubense. Chemical control is not economically effective and,therefore, breeding programs are necessary. Traditional field studies of new genotype resistance to this disease are time-consuming and destructive. Therefore,we developed a rapid and non-destructive procedure to differentiate field-grown banana resistant from susceptible clones. This procedure implicates application of culture filtrates of Fusarium oxysporum f. sp. cubense race 1 onto banana leaves. The relationship between duration of the fungal in vitro incubation, and the fungal culture fresh mass, the culture filtrate absorbency, and the Gross Michel (susceptible cultivar)leaf lesion area (after application of the culture filtrate) were similar and at 24day-incubation the highest values of the recorded indicators were observed. A comparison between Gross Michel and FHIA-01(resistant) was also performed. The most relevant differences between cultivars were observed at 48 hours after application of the culture filtrate, and in the middle-aged leaves. The position of the culture filtrate application in the leaf limb (distal, middle, proximal) was not determinant. A wider comparison among banana cultivars confirmed previous results informed by other researchers using different systems to study this plant-fungus interaction. Such a confirmation validates the effectiveness of the procedure described here to select rapid and non-destructively banana resistance to this disease at field level. This revised version was published online in August 2006 with corrections to the Cover Date.     

Potential of a Fusarium eumartii culture filtrate on the screening for wilting resistance in potato

Summary Fusarium solani f.sp. eumartii Carp. Snyder and Hansen (Fusarium eumartii) is a soil inhabitant that induces the so-called Potato Wilt and Stem End Rot disease. Prior to wilting, the pathogen induces peculiar small bronze spots on the leaflets. Failure to isolate F. eumartii from infected leaflets suggests the involvement of a toxin in the disease. The fungus was grown in liquid Richard's medium and thereafter a filtrate was obtained dialyzing (MW cutoff 12,000–14,000) and sterilizing the culture by filtration (0.22 m). Potato leaves treated with both the pathogen or the filtrate showed symptoms of bronze spots and significantly higher electrolyte leakage when compared to controls. Tomato leaves showed neither bronze spots nor electrolyte leakage after plant inoculation with the pathogen or with the filtrate treatment. Both, the absence of visible symptoms and the lack of electrolyte leakage in tomato could be associated to a certain degree of host specificity of the F. eumartii filtrate towards potato. The filtrate also induced symptoms similar to infections by F. eumartii in adult plants and in vitro plantlets of cultivars Huinkul MAG and Kennebec. Callus responses to the filtrate were related to responses of the cultivars to the pathogen in greenhouse. These results show the potential of the culture filtrate of F. eumartii for use in screening for wilting resistance.     

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.     

Selection parameters for resistance to Fusarium oxysporum f. sp. cubense race 1 and race 4 on diploid banana (Musa acuminata Colla)

Summary Shoot tip cultures from banana clones susceptible and resistant to Fusarium oxysporum f. sp. cubense (FOC) race 1 and race 4 were grown in vitro in the presence of different concentrations of fusaric acid and fungal crude filtrates or inoculated with a conidial suspension of FOC to assess correlation between in vivo and in vitro behaviour. Explants were susceptible to both filtrate and fusaric acid irrespective to their known field resistance/susceptibility response. No clear linkage between in vivo and in vitro behaviour was observed and our results suggest that the use of crude filtrate or non-host specific toxin (fusaric acid) in a screening programme for selecting a novel resistant genotype of Musa to FOC is not feasible. When peroxidase activity was used as a parameter to discriminate between sesceptibility and tolerance, results were in good agreement with field response of host plant to pathogens. Early enzymatic activity increased in the incompatible host-pathogen interaction but not in the compatible interaction.Abbreviations IBA Indolebutyric acid - 2iP 6-dimethylallylamino-purine - VCG Vegetative Compatibility Group - FOC Fusarium oxysporum f. sp. cubense - IEF isoelectrofocusing     

Phytotoxic effect of culture filtrate from Fusarium subglutinans the causal agent of fusariose of pineapple (Ananas comosus (L.) Merr

Two pineapple varieties differing in resistance to fusariose were examined for the phytotoxic effect of Fusarium subglutinans culture filtrate. The cultivars were Perolera (more resistant to pathovars of Fusarium subglutinans) and Smooth Cayenne (more susceptible). The phytotoxic effect of culture filtrate was assessed in tissue culture pineapple plantlets (by electrolyte leakage and placing the culture filtrate on wound leaf segments) and callus (inhibition of growth). Smooth Cayenne proved to be the most sensitive cultivar in each test, whereas Perolera showed resistance to the culture filtrate and its callus grew in the presence of high concentrations of culture filtrate that were completely toxic to Smooth Cayenne. These results show that plants can display cellular resistance to the culture filtrate. This revised version was published online in August 2006 with corrections to the Cover Date.     

Screening techniques and sources of resistance to root rots and wilts in cool season food legumes

Soilborne, fungal pathogens of cool season food legumes, including seed and seedling blights, rot rots, and wilts are described. Seed and seedling diseases are caused primarily by Pythium and Rhizoctonia spp. The most important fungi causing root rots include Aphanomyces euteiches, Fusarium solani, Pythium spp., Sclerotium rolfsii, and Macrophomina phaseolina. Wilt is caused primarily by various host-specific forms of Fusarium oxysporum. This paper discusses these diseases and screening procedures that emphasize standardization of inoculum levels, maintenance of virulent pathogen cultures, inoculum growth media, environmental conditions, and host plant age. Sources of resistance to these diseases are discussed.     

Induction of <Emphasis Type="Italic">Fusarium</Emphasis> wilt (<Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">pisi</Emphasis>) resistance in garden pea using induced mutagenesis and in vitro selection techniques

Wilt caused by Fusarium oxysporum f. sp. pisi is a serious production constraint for peas worldwide. An attempt was made to isolate wilt-resistant mutants in two susceptible pea genotypes, Arkel and Azad P-1, employing induced mutagenesis and in vitro selection techniques. Two thousand seeds of each genotype were mutagenized either with ethyl methane sulfonate (EMS, 0.2% and 0.3%) or gamma rays (5-22.5 kR) in ⁶⁰Co gamma cell for three consecutive years. Screening of different mutagenized populations under wilt-sick plots resulted in the isolation of 25 mutants exhibiting complete or enhanced wilt resistance compared to parental genotypes. Five of these wilt-resistant mutants also outperformed the susceptible background genotypes in terms of yield and other horticultural traits. Efforts were also made to isolate wilt-resistant regenerants from callus cultures exhibiting insensitivity to culture filtrate (CF) of F. oxysporum f. sp. pisi. A total of 250 regenerants (R ₀) were obtained from CF-insensitive calli selected from medium supplemented with 20% culture filtrate. When evaluated in artificially inoculated sick plots, only five R ₂ lines obtained from the regenerants exhibited enhanced wilt resistance compared to parental cultivars. However, the selected lines did not exhibit resistance levels equivalent to those shown by wilt-resistant lines isolated through in vivo mutagenesis. To conclude, induced mutagenesis through irradiation and EMS treatments exhibited superiority over in vitro selection for inducing wilt resistance in peas.     

Regeneration and genetic transformation of Spanish rice cultivars using mature embryos

To establish a plant regeneration system from embryogenic callus derived from mature rice embryos, the addition of aminoacids and the effect of two macronutrient solutions MSD and N6D to the basal callus induction medium was tested in three Spanish varieties, Senia, Tebre and Bahia. Aminoacids enhanced the production of embryogenic callus in Tebre and Senia whereas in the case of Bahia, embryogenic callus, which gave rise to a high rate of differentiated shoots, was induced without aminoacids. The macronutrient solution had also to be adjusted for each variety. Pre-regeneration treatment with ABA significantly improved the regeneration rate in all media tested, independently of the media in which the embryogenic callus were induced. In a comparison of growth regulators, BA yielded more shoots than Kin in all varieties whereas the effect of the auxins NAA or IAA was dependent on the variety. Transgenic plants from the three varieties were obtained via an Agrobacterium tumefaciens transformation system, using the optimised culture media. This revised version was published online in July 2006 with corrections to the Cover Date.     

Current status and future strategy in breeding pea to improve resistance to biotic and abiotic stresses

The economic importance and current progress made in studies of the host-parasite relationship and identification of sources of resistance and breeding strategies of some important biotic diseases of pea are reviewed in this paper. The root rot complex caused by Rhizoctonia solani, Fusarium solani, Aphanomyces euteiches, Pythium ultimum and Fusarium oxysporum f. sp. pisi, race 1 and 2 has been reported from all commercial pea growing areas of the world. Adequate sources of resistance have been identified and there has been impressive success in the control of the Fusarium wilt pathogen following the introduction of wilt-resistant cultivars. Leaf and stem diseases of pea caused by the Ascochyta complex, Peronospora viciae and Erysiphe pisi are prevalent in most temperate pea growing regions of the world. Several sources of resistance are available, some of which are surprisingly durable. The biochemical genetic parameters of phenolic content used for assaying resistance to Erysiphe pisi offers an alternative method of evaluating breeding material. Wild relatives of pea (Pisum fulvum and P. humile) are valuable additional sources of genetic variation and provide good sources of resistance to pests and diseases. In temperate rainfed pea growing areas of southern Australia, pea seed yield is more closely related to dry matter production than harvest index. Tall and leafy cultivars proved more productive than afila types.     

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 Response to Fusarium Elicitor and Toxic Substances in Crosses between Resistant and Susceptible Carnation Cultivars

To assess the correlation between in vivo resistance to F. oxysporum f. sp. dianthi and in vitro behaviour, calli from several resistant and susceptible Mediterranean carnation cultivars and F₁ progenies of crosses were tested both for the ability to grow on a medium containing crude culture filtrate of F. oxysporum and to produce phytoalexins following treatment with cell wall components (elicitor) of the same fungus. The results show no significant correlation between in vivo resistance and in vitro tolerance to culture filtrate of the fungus, while there was a good correlation in die case of phytoalexin production. Moreover, the character phytoalexin production behaved as a dominant in the crosses between a resistant and a susceptible cultivar.     

In vitro propagation of Japanese garden iris,Iris ensata Thunb.

Summary Explants of young scapes of Iris ensata were cultured on MS medium with 1 mg/l NAA, 1 mg/l 6-BA, 30 g/l sucrose and 10 g/l agar, and this species was characterized by high variety specificity for callus, shoot and root induction. Among 23 varieties and one wild form tested, Okichidori, Miyukisudare and Meiji-l exhibited a considerable rate of shoot induction, although these induced poorly rooted shoots. In addition, two types of callus induction such as green and white calli were observed, and the induction of green-type calli was significantly correlated with that of shoots. Surprisingly, the only modification, half-strength MS inorganic salts, for the above medium proved to be very effective for shoot induction in the scape culture. For shoots obtained from the scape culture, effects of sucrose concentrations and activated charcoal on root induction were examined by using 1/2 MS with 1 mg/l NAA, 1 mg/l 6-BA, 30 g/l sucrose and 10 g/l agar as the basic medium. The addition of 1% activated charcoal to the media had a marked effect for root induction independent of sucrose concentrations and varieties tested. The in vitro propagation technique of I. ensata is discussed.     

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.     

西瓜枯萎病菌的鉴定及申嗪霉素对其防效评价

由于重茬西瓜种植面积增多,西瓜枯萎病的发生面积逐步扩大,严重影响了西瓜生产。本研究用PDA培养基从中国农业科学院郑州果树研究所大棚温室萎蔫枯萎的西瓜上分离病原菌。形态学鉴定为尖孢镰刀菌;ITS序列分析表明其与西瓜枯萎病菌的序列一致性达到99%;发现该菌引起西瓜枯死严重,发病率达96.7%以上。因此,证实是西瓜枯萎病菌引起的。为了防治该病害,进行了温室和室内的申嗪霉素药效评价。室内抑菌实验和温室内药剂处理结果一致,1%申嗪霉素100倍防治效果最好,抑菌率达76.03%,灌根防治率达58.86%。因此,生产上建议在西瓜枯萎病发病早期使用1%申嗪霉素100倍连续3次灌根,每次间隔约8天左右,尽可能减少西瓜枯萎病菌对西瓜的影响。     

Localization ability,latent period and wilting rate in eleven carnation cultivars with partial resistance to Fusarium wilt

Summary The latent periods, wilting rates and percentages of diseased plants were analyzed for 11 carnation cultivars after root and after stem inoculation with race 2 of Fusarium oxysporum f.sp. dianth. There was no conclusive evidence for the presence of an independent extravascular resistance mechanism, except for Lena plants in which, additional to the vascular resistance components, independent root-bound factors causing retardation of the colonization and wilting process were found. A large variation was observed in the ablity of the cultivars to localize the pathogen in the vascular tissue shortly after infection of the xylem. This ability was positively correlated with the latent period, and negatively with the wilting rate and final disease index. In resistant cultivars, secondary compartmentalization of the fungus higher up in the stem was also observed. After stem inoculation, differences among the cultivars in localization ability and wilt-retarding actors could be identified at an early stage by comparing the precentages of non-colonized plants or the percentages of plants lacking vascular discolouration.     

In vitro culture of tissues,cells and protoplasts of Trifolium alexandrinum L. (Egyptian clover)

Summary Callus induction derived from root, hypocotyl and cotyledon explants were investigated for four cultivars of Trifolium alexandrinum L. The Murashige & Skoog (1962) medium containing 2.0 mg/L NAA and 0.5 mg/L BAP was able to induce callus from different explants. A wide range of culture media were tested to determine the morphogenetic potential of different callus types derived from different explant types. Shoot morphogenetic development was observed with the cultivars Sakha 4 and Giza 10. Cell suspension cultures were established from hypocotyl derived callus. Methods for isolation and culture of protoplasts from cotyledons are described.     

Screening techniques and sources of resistance to root diseases in cool season food legumes

Summary Soil-borne fungal diseases are among the most important factors, limiting the yield of grain legumes in many countries worldwide. Root rot, caused by Aphanomyces euteiches, Rhizoctonia solani, Fusarium solani and wilt, caused by several formae speciales of Fusarium oxysporum are the most destructive soil-borne diseases of pea, chickpea, lentil, fababean and lupin. The most effective control of these diseases is achieved through the use of resistant varieties. In this paper, recent advances in conventional and innovative screening methods for disease resistance are presented. Many grain legume accessions, which are maintained in national and international germplasm collections, have been evaluated for disease resistance and numerous resistant varieties have been released following incorporation of identified resistance genes from these sources. Recent identification of molecular markers tightly linked to resistance genes has greatly enhanced breeding programs by making marker assisted selection (MAS) possible and allowing the development of varieties with multiple disease resistance. Progress in the understanding of the biology of soil-borne fungal pathogens of grain legumes is also reviewed with particular reference to the genetic structure of their populations, diagnosis and host–pathogen interaction.     

The exploration of genetic resources of Portuguese cabbage and kale for resistance to several Brassica diseases

Summary Twenty three accessions of nine Portuguese cabbage and kale land races from different geographic origins were tested at the seedling stage for resistance to several important brassica diseases. Resistance to downy mildew (Peronospora parasitica), expressed as necrosis of the cotyledon mesophyll, was found in all the accessions. Type A resistance to cabbage yellows (Fusarium oxysporum f. sp. conglutinans race 1) was present in most of the landraces. Resistance to clubroot (Plasmodiophora brassicae race 6) was found in one accession of the Portuguese tree kale. High resistance to blackleg (Leptosphaeria maculans) and white rust (Albuco candida) was not detected, although several accessions showed 20 to 30% of plants with intermediate expression of resistance. All Portuguese cole accessions were susceptible to blackrot (Xanthomonas campestris pv. campestris).     

Increasing resistance to Fusarium crown and root rot in asparagus by gametophyte selection

In garden asparagus, Fusarium crown and root rot is the main cause of crop decline. Since chemical treatments are inefficient, efforts should focus on the development of resistant cultivars to control the disease. Toxic culture filtrate (TCF) of F. oxysporum has affected asparagus pollen germination and tube growth. Consequently, gametophyte selection was evaluated to ascertain if the application of selective agents at this level could increase selection efficiency. Two susceptible pistillate plants and one tolerant and one susceptible staminate plants were used in controlled crosses. Before pollination, a drop of a germination vehicle with TCF or without it was applied to the stigmas. Some pollinated pistils were fixed and analyzed by fluorescence microscopy; the rest were left on the plant for seed production. Fifty to 200 seeds were obtained per treatment combination (staminate plant x pistillate plant x pollination vehicle). The derived plantlets were inoculated in vitroand evaluated for disease symptoms. The application of TCF to stigmas reduced pollen germination and tube growth compared with untreated controls,regardless of the genotypic combination. Pollen germination and tube growth was poorer for the tolerant staminate genotype than for the susceptible one. When the TCF was applied, the number of seeds per pollination in comparison with the controls diminished only when the susceptible genotype was the pollinator. The percentage of affected root area of the progenies obtained after applying the TCF was lower than in the controls only when the tolerant genotype was the pollinator. Increasing Fusarium resistance in asparagus by means of gametophyte selection seems feasible. This revised version was published online in August 2006 with corrections to the Cover Date.     

In vitro culture of species and varieties of four Amaranthus L. species

Summary Callus induction, callus growth and organogenetic processes were investigated in hypocotyl and stem cultures of four species of Amaranthus each of which comprised several varieties. Callus formation occurred in almost all the varieties in 100% of explants. The combinations of naphtaleneacetic acid plus benzylaminopurine or 2,4-dichlorophenoxyacetic acid plus Kinetin were very effective in causing callus formation; the results for callus growth in presence of the same growth regulator combinations revealed in some cases significant differences among the varieties.As far organogenesis-based on a few varieties-only A. caudatus and A. hypochondriacus responded well forming shoots from callus when cultured in presence of indoleacetic acid plus Kinetin and/or indoleacetic acid plus benzylaminopurine. Root regeneration was also observed in several varieties. Complete plantlets were obtained from rooted shoots.The results are discussed in relation to hormonal effect and the genotype importance.