In vitro selection of yellow passion fruit genotypes for resistance to <Emphasis Type="Italic">Fusarium</Emphasis> vascular wilt

Fusarium vascular wilt (caused by Fusarium oxysporum f. sp. passiflorae) is a limiting factor in the cultivation of yellow passion fruit (Passiflora edulis). Since there is no effective and economically viable control available, development of resistant or at least tolerant cultivars are in demand. A number of procedures have been used for the initial selection of plant genotypes resistant to various fungal pathogens by means of a fungal culture filtrate or purified toxin. In this study, seeds and in vitro-grown plantlets of passion fruit were screened with different concentrations of either Fusarium oxysporum f. sp. passiflorae (FOP) culture filtrate (0, 20, 30, 40 or 50%, v/v) or fusaric acid (0.10, 0.20, 0.30 or 0.40 mM) supplemented in Murashige and Skoog (MS) basal media. Subsequently, selected plants were inoculated with a conidial suspension of FOP to assess correlation between in vivo and in vitro responses. In vitro sensitivity to the selective agents and the resistance response to the pathogen were also compared. Root growth was markedly influenced by FA, culture filtrate, and conidial suspension culture treatments. Observations indicated that roots were primary targets for attack by F. oxysporum. Successful in vitro selection of resistant genotypes by both FA and culture filtrate treatments suggested that this strategy was viable for accelerating breeding of passion fruit for resistance to the Fusarium vascular wilt.     

In vitro selection of barley and wheat for resistance against Helminthosporium sativum

Summary Calli derived from immature embryos of barley and wheat genotypes were screened for their resistance to purified culture filtrate produced by the fungus Helminthosporium sativum P.K. and B. Two selection methods were used: a continuous method in which four cycles of selection were performed one after another on toxic medium and a discontinuous method in which a pause on non-toxic medium was given after the second or third cycle of selection. The latter was superior as it allowed the calli to regain their regeneration ability. About 3,000 calli from two barley genotypes and 2,000 from two wheat genotypes were used for selection. The selection with the pathotoxins resulted in 6% to 17% surviving calli. Toxin tolerant callus lines of barley were characterised by protein isozymes. Zymograms showed one more isozyme than with the unselected sensitive callus. Barley and wheat plants have been regenerated from callus lines surviving the toxin treatment and in vivo testing against pathogen revealed that the majority of these plants were less sensitive.     

甘蔗赤腐病内生拮抗菌YC89的筛选及鉴定

【目的】筛选防治甘蔗赤腐病(sugarcane red rot)的生防菌株。【方法】实验以前期分离获得的甘蔗内生细菌为目标菌,以甘蔗赤腐病的病原真菌镰孢炭疽菌(Colletotrichum falcatum Went.)为指示菌,采用平板对峙法筛选对该病菌有较强抑制作用的菌株,然后通过琼脂扩散法测定菌株代谢产物对抑菌活性的影响,并对具有较好拮抗效果的高效菌株进行抑菌广谱性分析并对其进行鉴定。最后通过形态学、生理生化特征以及16SrDNA和gyrA序列分析对高效菌株YC89进行鉴定。【结果】经初筛筛选到抑菌带均大于1.60 cm的5株拮抗细菌,其中X22、W2、YC89抑菌带均高达1.87 cm。对初筛得到的5株内生菌进行复筛,结果所示菌株YC89、H1、X22、W2、YT93对镰孢炭疽菌的抑菌率都在75%以上,其中菌株YC89对该病菌的抑菌效果最好,其抑菌率为78%。菌株YC89的发酵液、上清液、过滤液及粗蛋白提取液对镰孢炭疽菌的生长有较强的抑制作用,且菌株YC89对玉米大斑病、甘蔗梢腐病、草莓灰霉病等7种病原菌也有较好的抑制效果。通过菌株鉴定结果,初步将YC89菌株鉴定为贝莱斯芽孢杆菌(Bacillus velezensis)。【结论】菌株YC89对镰孢炭疽菌具有较好的抑制效果,表明其对甘蔗赤腐病的生物防治具有较大的应用潜力。     

Selection of turmeric callus tolerant to culture filtrate of <Emphasis Type="Italic">Pythium Graminicolum</Emphasis> and regeneration of plants

Root rot disease tolerant clones of turmeric variety Suguna of Curcuma longa L. were isolated using continuous in vitro selection technique against pure culture filtrate of Pythium graminicolum. Large amount of profuse, compact, creamish white callus was obtained from in vivo vegetative bud when cultured on LSBM fortified with 2,4-D (3 mg l⁻¹) after 45 days of culture. Callus was challenged with pure culture filtrate of P. graminicolum to isolate viable callus within 30 days of culture, which was further subjected to pure culture filtrate treatment. After three cycles of treatment, four cell lines which are tolerant to culture filtrate was isolated through continuous in vitro selection and subcultured on regeneration medium LSBM fortified with BAP (4 mg l⁻¹) along with the control non-selected callus to obtain complete plantlets through discontinuous in vitro selection technique. Plants regenerated from tolerant and non-selected calli were screened for disease tolerance by adopting in vitro sick plot technique. The data obtained from this experiment revealed a ratio of 225:49 tolerant: susceptible in vitro clones retrieved from tolerant callus. However, plants regenerated from the CL1a₁ and non-selected calli were susceptible under in vitro sick plot technique. The root rot disease tolerant clones were hardened and established in soil with 90% survival frequency.     

Use of cell culture to screen sunflower germplasm for resistance to Phomopsis brown/gray stem spot

Resistance to Phomopsis sp. (Diaporthe sp.) brown/gray stem spot was confirmed by screening sunflower calli on shoot induction media amended with fungal filtrate. Calli of sunflower genotypes OCMS 74 and NS-H-45, which show resistance to the disease in field trials, remained viable on media with 15% (v/v) fungal filtrate, while calli of field susceptible genotypes RHA 273 and PAG/SF 103 were killed on media with as little as 7.5% fungal filtrate. Fresh weight of calli of all genotypes was significantly reduced by 2.5% fungal filtrate, and calli of all genotypes were killed by filtrate concentrations of 20%. These in vitro results corroborate prior field observations for disease reaction of these genotypes.     

Suppression of red rot caused by <Emphasis Type="Italic">Colletotrichum falcatum</Emphasis> on sugarcane plants using plant growth-promoting rhizobacteria

Bacterial strains with ability to suppress Colletotrichum falcatum were isolated from the rhizosphere of sugarcane. Thirty nine candidates, chosen on the basis of in vitro antagonism, inhibited C. falcatum growth by 15–65% on test plates. Twenty two isolates causing 50% or more in vitro inhibition were screened for their root colonization ability and biocontrol activity on micropropagated sugarcane plants under greenhouse conditions. Twelve strains suppressed red rot infection in plantlets, but no significant correlation was observed between in vitro pathogen inhibition and in vivo disease suppression. However, isolates showing root colonization over 5.2 log₁₀ CFU g⁻¹ of soil showed highest suppression of C. falcatum and reduction of red rot disease. Six strains with the capability to maintain a significant population in the sugarcane rhizosphere and with a high potential to control red rot were identified by 16S rDNA as Ochrobacterum intermedium NH-5, Pseudomonas putida NH-50, Bacillus subtilis NH-100, Bacillus subtilis NH-160, Bacillus sp NH-217 and Stenotrophomonas maltophilia NH-300.     

In vitro selection and regeneration of carnation (Dianthus caryophyllus L.) plants resistant to culture filtrate of Fusarium oxysporum f.sp. dianthi

Callus cultures derived from internodal segments of two cultivars of carnation susceptible to Fusarium oxysporum f.sp. dianthi were successfully used for in vitro selection for resistance to this pathogenic fungus. Resistant lines were selected by culturing calli on growth medium containing various concentrations of the culture filtrate of F. oxysporum f.sp. dianthi. Resistant calli obtained after two cycles (25 days/cycle) of selection were used for plant regeneration. About 32% of the plants regenerated from the resistant calli had acquired considerable resistance against the pathogen in the field. No phenotypic variation was observed in the selected regenerates.     

Bio-formulation of fluorescent Pseudomonas spp. induces systemic resistance against red rot disease and enhances commercial sugar yield in sugarcane

Abstract

Isolates of Pseudomonas spp. collected from the rhizosphere of sugarcane and cane stalks were screened for their antagonistic activity against Colletotrichum falcatum causing red rot disease in sugarcane. Talc formulations of the selected Pseudomonas spp. isolates improved the sugarcane vegetative sett germination and sugarcane growth under field conditions. Optimal talc formulations were assessed for their effect on induction of systemic resistance against the pathogen in the canes under artificial inoculation. All the four isolates CHAO, EP1, KKM1 and VPT4 were effective in inducing systemic resistance against C. falcatum in two seasons. In other studies, the bacterial formulations were assessed to induce resistance in sugarcane in a sick plot situation. In pathogen-infested soil the isolates KKM1 and CHAO suppressed the red rot disease development in susceptible sugarcane cultivar. Pseudomonas strains also protected sugarcane in a disease-endemic location. Pseudomonas spp treatment substantially improved the cane juice quality parameters affected by the pathogen infection. Standardization of talc formulations and application methods in the field offers potential for large-scale application of biocontrol formulations for the management of red rot disease in sugarcane growing regions.     

In vitro selection of resistant rice plants against rice blast caused by Pyricularia oryzae via tissue culture technique

Abstract

A step by step protocol for resistant calli selection via a tissue culture technique under stress of Pyricularia oryzae culture filtrates was followed. Rice embryos dissected apart from the endosperm of susceptible rice seeds (Giza 176 and Riho) to P. oryzae produced embryonic calli on media containing various growth regulators of 2,4-D at concentrations of 0, 1, 1.5 and 2 mg/L and/or benzyl amino purine (BAP) at 0, 0.5, 1 and 1.5 mg/L when incubated under complete dark conditions for three weeks. Embryonic explants only produced shoots on media containing BAP. Selection of resistant calli was carried out in vitro under the challenging stress of increasing concentration of the pathogen P. oryzae culture filtrate (CF) from “0” up to 100%. The selection protocol has two directions. The first is step-by-step selection from lower to higher selective (CF) concentrations. The second is the exchangeable continuous cycles with and without the same selective (CF) concentration until the end of the selection regime to avoid calli adaptation to (CF). The regenerated calli to plantlets occurred under (CF) stress showed resistance and susceptibility when exposed to the pathogen infection under greenhouse conditions. The results reveal that the resistance in regenerated rice plantlets to P. oryzae pathogen segregated as 1 resistant: 2 moderate resistant: 1 susceptible giving the predication that the resistance in rice to P. oryzae may be controlled by one pair of genes. The in vitro selective regime via tissue cultures is advisable for the selection of novel disease resistant plants because of its time saving, space, money, it is easily applied and has a bio-safe approach.     

Is the Phytophthora citrophthora culture filtrate a reliable tool for the in vitro selection of resistant Citrus variants?

Summary Nucellar calli from four Citrus cultivars with known resistance to the Phytophthora citrophthora pathogen were chosen as experimental material to test the pathogen's response to culture filtrate (CF). Sensitivity of the four calli to CF of the fungus was in reverse order to what is known on the susceptibility of the cultivars in vivo. Sensitivity of protoplasts derived from the same four calli to 2,4-dichlorophenoxyacetic acid (2,4-D) was in the same order as that of calli to CF. Protoplasts derived from calli selected for tolerance to CF showed a higher plating efficiency with increasing concentration of CF in the medium. TLC and GLC determinations showed the presence of indole acetic acid in the culture filtrate. Results indicate that CF of P. citrophthora cannot be used as a selection tool in vitro.Contribution No. 1655-E, 1986 series, from the Agricultural Research Organization, Bet-Dagan, Israel     

Molecular and Pathological Characterization of Colletotrichum falcatum Infecting Subtropical Indian Sugarcane

Red rot, caused by Colletotrichum falcatum, is the most significant problem of sugarcane worldwide. Pathological studies and three different marker systems were used to characterize 25 C. falcatum isolates collected from 18 subtropical sugarcane cultivars from 15 different sugarcane‐growing regions of three north‐eastern states of India to assess pathogen diversity. Of these 25 isolates, three were new (RR2A, RR15, RR83) from cultivars Co 7717, Co J83 and Co S88230, respectively, pathologically characterized on 13 standard differential hosts. Isolates Cf 01, Cf 08 and RR15 were the most, and Cf‐07 the least virulent. Molecular characterization using random amplified polymorphic DNA, universal rice primers (URP) and inter simple sequence repeat markers amplified a total of 161 alleles of which 159 were polymorphic (98.76%). Unweighted paired group method with arithmetic averages analysis of combined data of all the DNA markers obtained by three marker systems classified 25 isolates into six clusters at 34% genetic similarity with high Mantel matrix correlation (r = 0.83). The principal component analysis (PCA) of marker data explained 68% of the variation by first three components. Molecular diversity as revealed in these isolates is very high, but non‐structured. Isolate Co Pant 84212 was found to be genetically most diverse. We demonstrated for the first time that URPs derived from weed rice could successfully assess genetic diversity in C. falcatum. Molecular characterization of the C. falcatum isolates prevalent in north‐eastern India would enable red rot management strate‐gies, selection for resistance genes and development of resistant cultivars.     

利用辣椒疫霉培养滤液体外筛选胡椒抗瘟病无性系研究

在胡椒（Piper nigrum Linn．)茎尖丛生增殖技术的基础上,以印尼大叶种“Lampong Type”无菌实生苗作外植体源,利用辣椒疫霉(Phytophthora capsici)培养滤液对胡椒茎尖及其增殖形成的丛生芽进行体外选择。辣椒疫霉培养滤液的不同灭菌方法对辣椒疫霉培养滤液的毒性影响显著,过滤灭菌方式可以保持辣椒疫霉培养滤液的毒性,而高温高压灭菌方式则不能。随着辣椒疫霉培养滤液浓度的增加,茎尖和丛生芽的存活率和增殖率都在下降。在存活的茎尖或丛生芽培养中,一部分可正常增殖,其余的形成愈伤组织,或者保持生长停滞的休眠状态。在选择性培养基上继代培养2次后进行生根和移栽,利用离体叶片针刺接种法对温室条件下生长的移栽植株进行抗瘟病测定。以3次抗病检测均无明显症状的植株作为抗病株。随着辣椒疫霉培养滤液浓度的增加,得到的再生植株数量降低,但其中抗病株的比例提高。利用过滤灭菌方式加入选择性培养基的处理中,25％、50％和75％的辣椒疫霉培养滤液分别获得1株、4株和3株抗病株,分别占各处理再生植株总数的1．54％、20．00％和42．86％,共获得8株,占该组处理再生植株总数的8．70％。     

In vitro selection of alfalfa plants resistant to Fusarium oxysporum f. sp. medicaginis

Summary From two lines of Medicago sativa characterized by a high regeneration capability, calli resistant to culture filtrate of Fusarium oxysporum f. sp. medicaginis have been selected. In these calli regeneration capability was greatly reduced and only one plant per callus was recovered. Regenerated plants have been evaluated for resistance to culture filtrate and for in vivo resistance to the pathogen. Three plants out of eight were resistant to the fungus and a high correlation between resistance to culture filtrate and in vivo resistance was observed.Research work supported by C.N.R., Italy. Special grant I.P.R.A. Subproject 1, paper no. 1468     

In Vitro selection of groundnut cell lines fromCercosporidium personatum culture filtrates and regeneration of resistant plants through cell culture

Cell suspensions derived from immature leaves of the groundnut (Arachis hypogaea L.) were cultured in the presence and absence ofCercosporidium personatum pathotoxic culture filtrates. Cell viability and reactions of cell lines were determined after exposure to various concentrations (25–100%, v/v) of the filtrates. Cell lines have been selected for resistance to the toxin(s) produced byC. personatum. Selected cell lines were used for plant regeneration on regeneration media containingC. personatum culture filtrates. Plant regeneration frequency was found to be low in long-term cultures, whereas it was high in short-term cultures. The selfed progeny of the plants regenerated from the resistant cell lines showed resistance to the pathogen in the field. Six out of 82 plants exhibited enhanced resistance in the R₂ generation. The culture filtrate stimulated callus proliferation as well as plant regeneration at lower concentrations, a response that could prove to be very useful for obtaining disease resistant plants throughin vitro selection.     

Mechanism of resistance induced by plant activators against Colletotrichum falcatum in sugarcane

Abstract

A search for plant activators capable of inducing systemic resistance in sugarcane showed that plants pre-treated with synthetic signal inducers confer a high degree of resistance to Colletotrichum falcatum – the red rot pathogen. Among the various treatments, Acibenzolar S- methyl (ASM) was found to be very effective in restricting the pathogen colonization inside the inoculated cane stalk tissues. The induction of resistance was accompanied by a significant increase in peroxidases and polyphenoloxidases activities. A considerable decrease of pathogen titre in the pre-treated tissues as determined by ELISA, clearly demonstrated the restriction of pathogen colonization and proliferation in the sensitized cane stalks. Specific induction of new isoforms of peroxidases and polyphenoloxidases in C. falcatum elicitor treatment indicates the pathogen elicitor induced specific cellular response of sugarcane suspension-cultured cells.     

Somaclonal variants resistant to sugarcane mosaic virus and their agronomic characterization

Summary The sugarcane mosaic virus is a pathogen that causes severe disease to sugarcane. New varieties resistant to insects and pathogens have been developed in the last 70 yr through sugarcane breeding programs, but this takes between 10 and 15 yr. Tissue culture techniques are used as an aid for sugarcane improvement to increase desirable agronomic characteristics, such as disease resistance. In the present work, we report the generation of somaclonal variants from sugarcane (Saccharum spp.) cultivar PR62258 susceptible to Sugarcane Mosaic Virus, by somatic embryogenesis. These new variants identified as AT626 and BT627 are resistant to Sugarcanes Mosaic Virus strains A and B, respectively. We established an indirect enzyme linked inmunosorbent assay (ELISA) to test the presence of the viral particles in plants, and its was demonstrated that the leaves of resistant somaclones do not contain viral particles. The field performance of the somaclones AT626 and BT627 was similar to the field performance of the mother plant PR62258.     

Impact of cefotaxime on somatic embryogenesis and shoot regeneration in sugarcane

A cephalosporin antibiotic, cefotaxime (Omnatax™) promoted somatic embryogenesis and subsequent shoot regeneration in vitro from spindle in sugarcane irrespective of the genotypes as (CoJ 83, CoJ 88 and CoJ 64) culturered on MS medium with 2,4-D (2.5 mgl⁻¹) and kinetin (0.5 mgl⁻¹). Seven different concentrations of cefotaxime (100, 200, 300, 400, 500, 600 and 700 mgl⁻¹) were tested to find the optimal concentration of cefotaxime for somatic embryogenesis from callus cultures. Among the three varieties, calli of variety CoJ 83 incubated on MS medium with 2,4-D (2.5 mgl⁻¹) + kinetin (0.5 mgl⁻¹) + cefotaxime (500 mgl⁻¹) exhibited maximum somatic embryogenesis. To improve shoot regeneration, the callus was transferred to MS medium with BAP (0.5 mgl⁻¹) + kinetin (0.5 mgl⁻¹) in combination with different levels of cefotaxime. Highest frequency of shoot regeneration was observed in callus of CoJ 83 in the presence of 500 mgl⁻¹ cefotaxime. The plantlets could be successfully hardened in polybags and transferred to soil, where they exhibited normal growth. Our results convincingly demonstrated that cefotaxime improves somatic embryogenesis from spindle and regeneration from embryogenic calli of sugarcane and hence can be strongly recommended for rapid and large scale multiplication of sugarcane.Key words: Saccharum officinarum L., leaf segments, callus, plant regeneration, antibiotic     

Antifungal activity of chitinases produced by some fluorescent pseudomonads against Colletotrichum falcatum Went causing red rot disease in sugarcane

Chitinase production and growth of certain fluorescent pseudomonads isolated from sugarcane rhizosphere on different subtrates were studied. When chitin was substituted for glycerol in King's B medium, 3 of the 4 strains showed enhanced bacterial multiplication. Bacterial cells grown on chitin-containing medium showed enhanced antifungal activity against Colletotrichum falcatum Went causing red rot disease in sugarcane. Chitinase production was significantly higher when chitin was amended to King's B medium. Higher chitinase production was also recorded when fluorescent pseudomonad strains were grown in the medium containing crab-shell chitin. Cell-free bacterial culture filtrate from chitin-containing medium significantly inhibited mycelial growth of the pathogen. These cell-free conditioned media contained 3 to 7 polypeptides. Western blot analysis revealed five isoforms of chitinase with molecular masses of 47, 36, 32, 20 and 18.5 kDa. A possible role of chitinases in red rot disease management is discussed.     

In vitro selection of resistant/tolerant mutants lines of Citrus jambhiri Lush. using crude culture filtrate of Phytophthora parasitica and their randomly amplified polymorphic DNA analysis

This study deals with in vitro‐induced mutagenesis and selection of Phytophthora tolerant lines of Citrus jambhiri and their regeneration. For in vitro‐induced mutagenesis, cotyledons were treated with ethyl methane sulfonate (EMS) 100, 200 and 300 mm for different time durations viz. 1, 3, 6 and 9 hr. Callus cultures were established from EMS treated cotyledon explants on MS medium supplemented with 2.0 mg/L of 2,4‐D and 500 mg/L of malt extract. Calli derived from cotyledon were challenged in vitro on selective MS medium containing 5%–100% of culture filtrate (CF) of the Phytophthora parasitica. Selected mutagen‐treated calli showed resistance in vitro on media containing CF. Calli treated with 100 mm EMS for 6‐hr duration showed tolerance (24%) up to 75% CF after 4th selection cycle. While, calli treated with 200 mm for 6‐hr duration showed maximum tolerance (76%) up to 75% CF. Resistant calli were then transferred to MS regeneration medium for shoot bud regeneration. A dose‐dependent decrease in the regeneration capacity of the selected calli was observed with the increasing concentration of the CF. In randomly amplified polymorphic DNA analysis, plantlets showed different banding pattern in comparison with the control plant, which confirms the presence of variations at genetic level.     

Somacional variation and eyespot toxin tolerance in sugarcane

The analysis of sugarcane plants regenerated from culture for their reaction to eyespot (Helminthosporium sacchari) toxin is described. A total of 480 culture-derived plants (somaclones) from cultivar Q101 were characterized. Some of these plants derived from cultures which had been subjected to selection with the eyespot toxin and others were derived without overt selection. Leaves were assayed for their toxin reaction. A very high frequency of toxin-tolerant variants was found. The distribution was even further biased toward resistance in those plants regenerated from cultures exposed to toxin selection.A total of 85 somaclones was analysed for the stability of their increased toxin tolerance to the primary somaclone; 22% were more tolerant; 38% were more susceptible. These results are discussed as they relate to the possibility of using consecutive vegetative segregation.Six tolerant variants were also passed through a second tissue culture cycle and 60 secondary somaclones were assayed. Twenty four (40%) of these plants had a similar tolerance to the primary somacione; 22% were more tolerant; 38% were more susceptible. These results are discussed as they relative to the possibility of using consecutive cycles of culture to stack improved characters into a sugarcane cultivar.