Assessing Quantitative Resistance against Leptosphaeria maculans (Phoma Stem Canker) in Brassica napus (Oilseed Rape) in Young Plants

Quantitative resistance against Leptosphaeria maculans in Brassica napus is difficult to assess in young plants due to the long period of symptomless growth of the pathogen from the appearance of leaf lesions to the appearance of canker symptoms on the stem. By using doubled haploid (DH) lines A30 (susceptible) and C119 (with quantitative resistance), quantitative resistance against L. maculans was assessed in young plants in controlled environments at two stages: stage 1, growth of the pathogen along leaf veins/petioles towards the stem by leaf lamina inoculation; stage 2, growth in stem tissues to produce stem canker symptoms by leaf petiole inoculation. Two types of inoculum (ascospores; conidia) and three assessment methods (extent of visible necrosis; symptomless pathogen growth visualised using the GFP reporter gene; amount of pathogen DNA quantified by PCR) were used. In stage 1 assessments, significant differences were observed between lines A30 and C119 in area of leaf lesions, distance grown along veins/petioles assessed by visible necrosis or by viewing GFP and amount of L. maculans DNA in leaf petioles. In stage 2 assessments, significant differences were observed between lines A30 and C119 in severity of stem canker and amount of L. maculans DNA in stem tissues. GFP-labelled L. maculans spread more quickly from the stem cortex to the stem pith in A30 than in C119. Stem canker symptoms were produced more rapidly by using ascospore inoculum than by using conidial inoculum. These results suggest that quantitative resistance against L. maculans in B. napus can be assessed in young plants in controlled conditions. Development of methods to phenotype quantitative resistance against plant pathogens in young plants in controlled environments will help identification of stable quantitative resistance for control of crop diseases.     

Comparative physiological responses of the yeast halotolerance genes expressed in transgenic lines of tomato cv Rio Grande under saline conditions

We previously analyzed the transgenic lines of tomato cv Rio Grande over-expressing the yeast HAL I and HAL II genes for their response to salt stress under in vitro conditions. In this study, six homozygous tomato lines harbouring the yeast HAL I or HAL II genes with highest expression level were selected for exploring their physiological responses against different salt stresses in the field. These transgenic plants showed significant growth and improved water content in comparison with control under 100 and 150 mM salt stress conditions. The HAL I and HAL II lines showed better Ca²⁺ content than their control counterparts. Furthermore, the transgenic lines exhibited lower values of relative electrical conductivity and improved resistance against the fungal pathogens Fusarium oxysporum and Alternaria solani when tested by detached leaf and agar tube dilution assays. Physiological analyses carried out in this study suggest an involvement of multiple mechanisms in transgenic tomato plants harbouring yeast genes to confer biotic and abiotic tolerance under stress conditions.     

Development of interspecific Solanum lycopersicum and screening for Tospovirus resistance

Tospovirus has emerged as a serious viral pathogen for several crops including tomato. The tomato production is being severely affected worldwide by Tospovirus. Some reports have been published about the association of plant virus and development of human disease either by direct or indirect consumption. Resistance to this virus has been identified as good source in wild tomato species (Lycopersicum peruvianum). But the introgression of resistance genes into cultivated tomato lines and the development of interspecific hybrid are hampered due to incompatibility, fertilization barriers and embryo abortion. But this barrier has been broken by applying the embryo rescue methods. This study describes the development of interspecific hybrid tomato plants by highly efficient embryo rescue method and screening for Tospovirus resistance. The interspecific hybrid tomato plants were developed by making a cross between wild tomato species (L. peruvianum) and cultivated tomato (Solanum lycopersicum). The immature embryos were cultured in standardized medium and interspecific hybrids were developed from embryogenic callus. The immature embryos excised from 7 to 35 days old fruits were used for embryo rescue and 31 days old embryos showed very good germination capabilities and produced the highest number of plants. Developed plants were hardened enough and shifted to green house. The hybrid nature of interspecific plants was further confirmed by comparing the morphological characters from their parents. The F1, F2 and F3 plants were found to have varying characters especially for leaf type, color of stem, fruits, size, shapes and they were further screened for virus resistance both in lab and open field followed by Enzyme linked Immunosorbant Assay confirmation. Finally, a total of 11 resistant plants were selected bearing red color fruits with desired shape and size.     

The Effects of Root-knot Nematode Infection and Mi-mediated Nematode Resistance in Tomato on Plant Fitness

The Mi-1.2 resistance gene in tomato (Solanum lycopersicum) confers resistance against several species of root-knot nematodes (Meloidogyne spp.). This study examined the impact of M. javanica on the reproductive fitness of near-isogenic tomato cultivars with and without Mi-1.2 under field and greenhouse conditions. Surprisingly, neither nematode inoculation or host plant resistance impacted the yield of mature fruits in field microplots (inoculum=8,000 eggs/plant), or fruit or seed production in a follow-up greenhouse bioassay conducted with a higher inoculum level (20,000 eggs/plant). However, under heavy nematode pressure (200,000 eggs/plant), greenhouse-grown plants carrying Mi-1.2 had more than ten-fold greater fruit production than susceptible plants and nearly forty-fold greater estimated lifetime seed production, confirming prior reports of the benefits of Mi-1.2. In all cases Mi-mediated resistance significantly reduced nematode reproduction. These results indicated that tomato can utilize tolerance mechanisms to compensate for moderate levels of nematode infection, but that the Mi-1.2 resistance gene confers a dramatic fitness benefit under heavy nematode pressure. No significant cost of resistance was detected in the absence of nematode infection.     

Effects of inoculum concentration, leaf age and wetness period on the development of dark leaf and pod spot (Alternaria brassicae) on oilseed rape (Brassica napus)

Experiments were done under controlled environment and glasshouse conditions to study the effects of inoculum concentration, leaf age and wetness period on the development of dark leaf and pod spot (Alternaria brussicae) on oilseed rape (Brassica napus). On leaves of potted oilseed rape plants (cv. Bienvenu) inoculated with A. brassicae conidial suspensions, the severity (number of lesions cm^-2) of dark leaf spot increased as inoculum concentration increased from 80 to 660 spores ml^-1and as leaf age increased from 4 to 14 days. On pods on detached racemes of spring oilseed rape (cv. Starlight), the incidence of dark pod spot (% of pods diseased) increased as inoculum concentration increased from 80 to 10⁴spores ml^-1. Increasing inoculum concentration above 10⁴spores ml^-1did not increase the incidence but did increase the severity of dark pod spot. A minimum wetness period of 4 h was needed for infection of oilseed rape leaves (cv. Envol) by A. brussicue at 18°C and disease severity increased with increasing wetness period up to 12 h. The length of dry interruptions after 3–8 h of initial wetness affected the severity of dark leaf spot. A second wetness period increased the severity of dark leaf spot if the dry interruption was ≤ 6 h and if the first wetness period was ≤ 8 h. The incubation period of A. brassicae decreased from 3.5 to 2.5 days as inoculum concentration increased from 80 to 660 spores ml^-on leaves (cv. Bienvenu) at 17–25°C and from 3.8 to 1.0 day as inoculum concentration increased from 80 to ≥2 ≥ 10³spores ml^-1on pods (cv. Starlight) at 18°C.     

Leaf disc transformation of cultivated tomato (L. esculentum) using Agrobacterium tumefaciens

The leaf disc transformation/regeneration system was modified for tomato (L. esculentum). Both leaf explants and cotyledon/hypocotyl sections can be used to regenerate transformed plants. We have obtained over 300 transgenic plants from eight tomato cultivars. We have evidence for both single and multi-copy insertions of the T-DNA, and have demonstrated inheritance of the T-DNA insert in the expected Mendelian ratios. Several heterologous promoters function in tomato. A reduced efficiency of transformation was observed with binary T-DNA vectors as compared to co-integrate T-DNA vectors. The ease of the leaf disc method makes tomato a premier experimental organism for plant biotechnology.Abbreviations BA benzyl adenine - IAA indole acetic acid - LB Luria Broth     

Distribution of acid invertase in the tomato plant

Acid invertase activity in Lycopersicon esculentum was highest in the locular wall of ripe fruit and lowest in roots. Activity was greater in leaf laminae than in petiole tissue and increased with leaf age, whereas there was more invertase in the upper part of the stem compared with the older portion. Activity in whole fruit increased with increasing ripeness and was greatest in overripe fruit. Of various tissues from a number of wild tomato species examined, the fruit of L. pimpinellifolium were particularly rich in the enzyme, in contrast to the fruit of L. hirsutum, L. hirsutum, var. glabratum and L. peruvianum which had low activity.     

Induced Resistance to Meloidogyne hapla by other Meloidogyne species on Tomato and Pyrethrum Plants

Advance inoculation of the tomato cv. Celebrity or the pyrethrum clone 223 with host-incompatible Meloidogyne incognita or M. javanica elicited induced resistance to host-compatible M. hapla in pot and field experiments. Induced resistance increased with the length of the time between inoculations and with the population density of the induction inoculum. Optimum interval before challenge inoculation, or population density of inoculum for inducing resistance, was 10 days, or 5,000 infective nematodes per 500-cm³ pot. The induced resistance suppressed population increase of M. hapla by 84% on potted tomato, 72% on potted pyrethrum, and 55% on field-grown pyrethrum seedlings, relative to unprotected treatments. Pyrethrum seedlings inoculated with M. javanica 10 days before infection with M. hapla were not stunted, whereas those that did not receive the advance inoculum were stunted 33% in pots and 36% in field plots. The results indicated that advance infection of plants with incompatible or mildly virulent nematode species induced resistance to normally compatible nematodes and that the induced resistance response may have potential as a biological control method for plant nematodes.     

水稻铁害和应激乙烯释放的关系

在热带地区的水稻栽培中,常遇到水稻青铜病（ｂｒｏｎｚｉｎｇ）的危害．已知它是由水田中高浓度的亚铁离子所引起,故又叫铁害．但至今没有可靠的生理诊断指标用于抗性品种的筛选．本文研究了铁害与应激乙烯释放的关系,试图以应激乙烯的释放作为铁害的生理诊断指标．试验用两种方法模拟水稻致病．第一种方法是将水稻离体叶片的剪口端浸入ＦｅＳＯ４溶液中,靠叶片蒸腾作用吸收Ｆｅ＋＋而致病．另一种方法是在水培培养液中加入ＦｅＳＯ４通过水稻根系吸收Ｆｅ＋＋而致病．研究结果表明,当处理离体叶片时,发病强度和应激乙烯释放量呈显著相关,但叶片内铁含量的增加与发病强度和应激乙烯释放都没有相关性．而处理完全植株时,叶片中乙烯释放几乎不受影响．当部分或全部切除根时,叶片中乙烯释放则可被亚铁离子激发。表明水稻根系限制了Ｆｅ＋＋的吸收速率,而Ｆｅ＋＋进入叶组织的速率又决定应激乙烯的释放和组织的伤害程度．因此,叶片应激乙烯的释放作为铁害的生理诊断指标只有在当根系受到某种伤害时才可能适用,譬如移栽和毒性土壤等因素造成的根系的伤害．     

Resistenz verschiedener Kultur- und Wildtomatenpflanzen (Lycopersicon spp.) gegenüber Botrytis cinerea Pers.

Resistance of different cultivated and wild tomato plants (Lycopersicon spp.) to Botrytis cinerea Pers. 20 provenances of different cultivated and wild tomato plants (Lycopersicon spp.) were screened for resistance to Botrytis cinerea Pers. using an in vitro-leaf necrosis test. The Botrytis resistance decreased with increasing age of the leaves corresponding to their insertion height (relative youth resistance respectively senescence susceptibility). The 6 B. cinerea-isolates used for inoculation differed significantly in virulence. With increasing inoculum age a virulence reduction of the various B. cinerea-isolates occurred. Within the investigated test plant collection 2 wild species –L. columbianum and L. hirsutum– proved to be resistant in each stage of development to all B. cinerea-isolates and additionally showed field resistance.     

The effect of early blight disease caused by Alternaria solani on shoot growth of young tomato plants

The effect of increasing spore concentration of Alternaria solani (Early blight disease) on the shoot growth of young tomato plants was analysed. Changes in growth were related to the severity of infection which increased with increasing inoculum. Leaf production was not affected but dry weights and especially leaf expansion were decreased. The effective leaf areas of the five inoculated leaves (L1-L5 numbered from the plant base) were drastically decreased by expanding necrotic lesions and, to a lesser extent, by premature leaf fall. Healthy leaves expanding soon after inoculation (L6, L7) were markedly affected by the disease on the lower leaves and had decreased specific leaf areas (ratio of leaf area to leaf dry weight) but later formed (from L8) leaves were less affected and had greater specific leaf areas than equivalent leaves on uninoculated plants.     

Tomato plant and leaf age effects on the probing and settling behavior of Frankliniella fusca and Frankliniella occidentalis (Thysanoptera: Thripidae)

The effect of tomato, Solanum lycopersicum L., plant and leaf age on the probing and settling behavior of Frankliniella fusca (Hinds) and F. occidentalis (Pergande) was studied using electrical penetration graph technique and whole plant bioassays. Male and female F. fusca probed and ingested more and for longer periods of time on 3- and 4-wk-old plants compared with 6- and 8-wk-old plants. Female F. fusca probed and ingested more frequently than males in the plant age experiment, but not in the leaf age experiment. F. fusca probed and ingested more frequently on 2- and 4-wk-old leaves compared with 1-wk-old leaves. Plant age did not affect the probing frequency or duration of F. occidentalis; however, males probed and ingested longer than females in the plant age experiment and on the oldest leaf in the leaf age experiment. Both thrips species preferred to settle on 3-wk-old plants. F. fusca preferred to settle on 4-wk-old leaves after settling randomly for an hour. F. occidentalis showed no settling preference relative to leaf age. The preference of F. fusca for young plants suggests that this species could attack tomato plants at a very early stage, which is important for understanding its role as a vector in the transmission of Tospovirus in the field.     

Analysis of salinity effects on basil leaf surface area, photosynthetic activity, and growth

Basil (Ocimum basilicum L.) seedlings were cultured on liquid medium in controlled conditions. Two varieties differing in leaf size were compared. When plants were 30?days old, the medium was supplemented with 50?mM NaCl. After 15?days of treatment, root, stem and leaf biomass, leaf number, and leaf surface area were measured. Ion accumulation was determined in roots, stems, and leaves. Photosynthetic parameters (CO₂ fixation rate, internal CO₂ concentration, stomatal conductance) as well as transpiration rate were determined on separate leaves. Electrolyte leakage and malondialdehyde content were used to estimate damage to membranes and lipid peroxidation, respectively. Several antioxidant enzymatic activities were used as proxies of oxidative stress. High Na⁺ concentration was reached in leaf tissues. Salt restricted whole plant biomass deposition rate by diminishing leaf number and leaf expansion, as well as photosynthetic activity were estimated from whole plant biomass production per unit leaf surface area. Diminished stomatal conductance restricted CO₂ fixation rate, and decrease in chlorophyll content presumably limited photosynthetic activity. Lipid peroxidation revealed damages to membranes. The magnitude of these responses differed between the two varieties, indicating that an intraspecific variability in salt response exists in basil.     

Survival of inoculum of the leaf blight fungus Phytophthora colocasiae infecting taro, Colocasia esculenta in the Solomon Islands

Phytophthora colocasiae was successfully isolated by baiting with detergent-treated taro leaf discs 8 cm diameter placed on water slurries of soil, on suspensions of macerated infected leaf lesions or on the washings from petioles of harvested plants. Taro root tips, detached or left on corms, were not susceptible to zoospores of P. colocasiae nor were detached root tips of Lupinus angustifolius. Cubes of taro corm used as baits, and agar selective for Phycomycetes which was inoculated directly with soil, both became too heavily overrun by Phythium splendens to allow detection of P. colocasiae. Investigations indicated that inoculum on lesions of detached leaves and in soil remains viable for only a few days. Petiole bases which comprise the bulk of the ‘tops’ used for vegetative propagation, lost detectable natural inoculum rapidly (2 days) if stored dry, but less rapidly (14 days) if planted immediately in the field. Artifically augmenting surface inoculum with naturally produced sporangia considerably extended the periods of detectability, probably by increasing the chances that a few propagules would survive, especially during dry storage. Incubation of inoculated tops in high humidity led to active infection and sporulation on petioles, especially on cut ends, a situation that might be paralleled under suitable moisture conditions in the field. Of several aroid species tested by artificial inoculation only Alocasia macrorrhiza was susceptible. Natural infection of this plant has not been seen, making it an unlikely alternate host of P. colocasiae under field conditions. Thus perennation between taro crops is effected by shortlived surface propagules and possibly also by mycelium within lesions on petioles. Reduction of the former and prevention of the latter might be achieved by dry storage of tops for 2 to 3 wk.     

Plant Resistance to the Moth <Emphasis Type="Italic">Tuta absoluta</Emphasis> (Meyrick) (Lepidoptera:Gelechiidae) in Tomato Cultivars

The resistance of 11 tomato cultivars (Ps-6515, Berlina, Poolad, Petoprid-5, Zaman, Matin, Golsar, Sandokan-F1, Golshan-616, Sadeen-95 and Sadeen-21) to the tomato moth, Tuta absoluta (Meyrick) (Lepidoptera:Gelechiidae) was investigated under field conditions. A randomized complete block design was used with three replications. Data analysis indicated that there were significant differences (P?<?0.05) among cultivars regarding leaflet damage, leaf damage, overall plant damage, number of mines per leaf, number of holes on the stem, and fruit. Our findings revealed that the cultivars Berlina, Golsar, Poolad, and Zaman were less suitable cultivars, suggesting that they are more resistant to the tomato moth than the other cultivars. The high density of leaf trichomes present in the cultivars Berlina, Zaman, and Golsar can be one of the possible causes of resistance to T. absoluta. Knowledge of the extent of susceptibility or resistance of cultivars to a pest on a crop is one of the fundamental components of integrated pest management (IPM) programs for any crop.     

Comparison of Methods for Assessing Resistance to Meloidogyne arenaria in Peanut

Use of resistant cultivars is a desirable approach to manage the peanut root-knot nematode (Meloidogyne arenaria). To incorporate resistance into commercially acceptable cultivars requires reliable, efficient screening methods. To optimize the resistance screening protocol, a series of greenhouse tests were done using seven genotypes with three levels of resistance to M. arenaria. The three resistance levels could be separated based on gall indices as early as two weeks after inoculation (WAI) using 8,000 eggs of M. arenaria per plant, while four or more weeks were needed when 1,000–6,000 eggs/plant were used. High inoculum densities (over 8,000 eggs/plant) were needed to separate the three resistance levels based on eggs per gram of root within eight WAI. A gall index based on percentage of galled roots could separate the three resistance levels at lower inoculum levels and earlier harvest dates than other assessment methods. The use of eggs vs. second-stage juveniles (J2) as inoculum provided similar results; however, it took three to five more days to collect J2 than to collect eggs from roots. Plant age affected gall index and nematode reproduction on peanut, especially on the susceptible genotypes AT201 and D098. The genotypes were separated into their correct resistance classes when inoculated 10 to 30 days after planting, but were not separated correctly when inoculated on day 40.     

Response of tomato genotypes to bacterial speck (Pseudomonas syringae pv. tomato)

Two genotypes of tomato A 100 and Ontario 7710 which were inoculated separately with four strains of Pseudomonas syringae pv. tomato differed significantly in disease severity (susceptibility) to bacterial speck. At both concentrations of inoculum of each strain used (10⁷ and 10⁸ cfu/ml) A 100 appeared to be highly susceptible whereas Ontario 7710 showed very low or no susceptibility. The significant differences in virulence between strains and in response of tomato plants in three replicate experiments were found. Generally, concentration of inoculum 10⁷ cfu/ml was too low to induce consistent level of disease severity. The obtained results indicate the importance of consistent and favorable conditions for disease development in screening of tomato resistance to bacterial speck.     

Partial purification and characterization of 2, 4-diacetylphloroglucinol producing Pseudomonas fluorescens VSMKU3054 against bacterial wilt disease of tomato

We find out the antimicrobial potential of partially purified 2,4-diacetylphloroglucinol (DAPG) against Ralstonia solanacearum and fungal plant pathogens isolated from tomato rhizobacterium Pseudomonas fluorescens VSMKU3054. The present study is mainly focused on the control of wilt disease of tomato by our isolate VSMKU3054 and DAPG. The cell free culture filtrate of P. fluorescens VSMKU3054 was significantly arrested the growth of R. solanacearum and fungal pathogens such as Rhizoctonia solani, Sclerotium rolfsii, Macrophomina phaseolina and Fusarium oxysporum compared to control. The existence of DAPG from the crude metabolites of P. fluorescens VSMKU3054 was confirmed on TLC with Rf value 0.34, which is coincide with that of authentic phloroglucinol. The partially purified DAPG exhibited much higher activity against R. solanacearum at 30 µg/ml than the fungal plant pathogens compared to control. The antimicrobial partially purified compound was identified as DAPG by UV, FT-IR and GC–MS analysis. The percentage of live cells of R. solanacearum when supplemented with DAPG at 30 µg/ml, significantly controlled the living nature of R. solanacearum up to 68% compared to tetracycline and universal control observed under high content screening analysis. The selected isolate P. fluorescens VSMKU3054 and DAPG significantly controlled wilt disease of tomato up to 59.5% and 42.12% on 3rd and 7th days compared to positive and negative control by detached leaf assay. Further, in silico analysis revealed that high interaction of DAPG encoding protease with lectin which is associated with R. solanacearum. Based on our findings, we confirmed that P. fluorescens VSMKU3054 and DAPG could be used a potential bio inoculants for the management of bacterial wilt disease of tomato.     

Glucosinolate content and nematicidal activity of Brazilian wild mustard tissues against Meloidogyne incognita in tomato

The wild mustard (Brassica juncea L.), an invasive weed of winter crops in Brazil, was evaluated for glucosinolate content of its plant tissues and nematicidal activity of its dry leaf meal (LM), whole seed meal (WSM) and hexane defatted seed meal (DSM) against Meloidogyne incognita on tomato plants. Sinigrin was the major glucosinolate in LM, WSM and DSM, occurring at concentration of 0.11, 12.2 and 21.9 mg/gdw, respectively. Allyl isothiocyanate (AITC) was the major degradation product and its concentration was highest in DSM followed by WSM and LM. The number of galls, egg masses and eggs on tomato plants was reduced by over 90% by amending soil with 1.6% LM, 0.2% WSM, or 0.05% DSM. Exposure to the volatiles from the amended soils reduced egg eclosion. The soil amendment with LM, WSM and DSM killed the second stage juveniles of M. javanica, M. enterolobii (=M. mayaguensis) and Heterodera glycines. The efficacy of the LM, WSM and DSM for nematode suppression was related to the amount of AITC released in soil.     

Detached leaf assay to screen for host plant resistance to Helicoverpa armigera

The noctuid Helicoverpa armigera (Hübner) is a major insect pest of chickpea Cicer arietinum L., pigeonpea Cajanus cajan (L.) Millsp., peanut Arachis hypogaea L., and cotton Gossypium spp., and host plant resistance is an important component for managing this pest in different crops. Because of variations in insect density and staggered flowering of the test material, it is difficult to identify cultivars with stable resistance to H. armigera across seasons and locations. To overcome these problems, we standardized the detached leaf assay to screen for resistance to this pest in chickpea, pigeonpea, peanut, and cotton under uniform insect pressure under laboratory conditions. Terminal branch (three to four fully expanded leaves) of chickpea, first fully expanded leaf of cotton, trifoliate of pigeonpea, or quadrifoliate of peanut, embedded in 3% agar-agar in a plastic cup/jar of appropriate size (250-500-ml capacity) infested with 10-20 neonate larvae can be used to screen for resistance to H. armigera. This technique keeps the leaves in a turgid condition for approximately 1 wk. The experiments can be terminated when the larvae have caused > 80% leaf damage in the susceptible check or when differences in leaf feeding between the resistant and susceptible checks are maximum. Detached leaf assay can be used as a rapid screening technique to evaluate germplasm, segregating breeding materials, and mapping populations for resistance to H. armigera in a short span of time with minimal cost, and under uniform insect infestation. It also provides useful information on antibiosis component of resistance to the target insect pest.