Delayed Symptom Development Research Articles

Transgenic tobacco plants expressing the coat protein of cucumber mosaic virus show different virus resistance

Transgenic tobacco (Nicotiana tabacum cv. Xanthi-nc) plants were regenerated after cocultivation of leaf explants withAgrobacterium tumefaciens strain LBA4404 harboring a plasmid that contained the coat protein (CP) gene of cucumber mosaic virus (CMV-As). PCR and Southern blot analyses revealed that the CMV CP gene was successfully introduced into the genomic DNA of the transgenic tobacco plants. Transgenic plants (CP+) expressing CP were obtained and used for screening the virus resistance. They could be categorized into three types after inoculation with the virus: virus-resistant, delay of symptom development, and susceptible type. Most of the CP+ transgenic tobacco plants failed to develop symptoms or showed systemic symptom development delayed for 5 to 42 days as compared to those of nontransgenic control plants after challenged with the same virus. However, some CP+ transgenic plants were highly susceptible after inoculation with the virus. Our results suggest that the CP-mediated viral resistance is readily applicable to CMV disease in other crops.

Forced evolution reveals the importance of short open reading frame A and secondary structure in the cauliflower mosaic virus 35S RNA leader.

Cauliflower mosaic virus pregenomic 35S RNA begins with a long leader sequence containing an extensive secondary structure and up to nine short open reading frames (sORFs), 2 to 35 codons in length. To test whether any of these sORFs are required for virus viability, their start codons were mutated either individually or in various combinations. The resulting viral mutants were tested for infectivity on mechanically inoculated turnip plants. Viable mutants were passaged several times, and the stability of the introduced mutations was analyzed by PCR amplification and sequencing. Mutations at the 5'-proximal sORF A and in the center of the leader resulted in delayed symptom development and in the appearance of revertants. In the central leader region, the predicted secondary structure, rather than the sORF organization, was restored, while true reversions or second-site substitutions in response to mutations of sORF A restored this sORF. Involvement of sORF A and secondary structure of the leader in the virus replication cycle, and especially in translation of the 35S RNA via ribosome shunting, is discussed.

Geminivirus Resistance in Transgenic Tobacco Expressing Mutated BC1 Protein

Tobacco explants were transformed by Agrobacterium-mediated transformation with sense and antisense constructs of the movement protein genes (BC1 and BV1) of tomato mottle geminivirus (TMoV). Transgenic plants were tested for virus resistance either by exposure to viruliferous whiteflies carrying TMoV or cabbage leaf curl geminivirus (CabLCV) for a 72-h inoculation period or by continuous exposure to TMoV during the life of the plants. The transgenic lines were scored for disease symptoms, and virus replication and distribution were determined by enzyme-linked immunosorbent assay and dot blot hybridizations. Transgenic plants which expressed a mutated form (identified in a previous study) of the BC1 gene showed TMoV and CabLCV resistance. Three resistant phenotypes were observed: a delay in symptom development, a recovery from early symptoms, and an absence of virus symptoms at all stages. Geminivirus was detected in inoculated leaves but was not readily detected in leaves beyond the inoculation sites in the highly resistant plants. The geminivirus resistance conferred by expression of the spontaneously mutated BC1 gene (detected after transformation) in transgenic tobacco may involve transdominant negative interference.

Hemibiotrophic infection of round-leaved mallow by Colletotrichum gloeosporioides f. sp. malvae in relation to leaf senescence and reducing reagents

The infection of round-leaved mallow ( Malva pusilla ) leaves by Colletotrichum gloeosporioides f. sp. malvae was studied using light and confocal microscopy. Conidia germinated and produced appressoria within 24 h after inoculation. An infection peg arose from the base of the appressorium and directly penetrated an epidermal cell. An intracellular infection vesicle appeared beneath the penetration site by 48 h after inoculation. Large primary hyphae (LPH, approx. 4 μ diam.) emerged from the vesicle and grew intracellularly through several adjacent epidermal cells and then intercellularly between mesophyll cells. Epidermal cells infected by LPH maintained their viability, as shown by their ability to plasmolyse and accumulate neutral red stain. No visible disease symptoms appeared during this biotrophic stage of infection. Thin secondary hyphae (TSH, approx. 2 μ diam.) developed from LPH in 4–5 days after inoculation and were associated with the appearance of necrotic lesions. Host cell wall maceration was visible only during the necrotrophic stage. The duration of the biotrophic stage decreased as mallow leaves became older or were senesced by placing them in the dark. TSH and host cell necrosis developed as soon as 48 h after inoculation of the most senescent leaves, and TSH were observed directly emerging from the infection vesicle. Application of thiol reagents, such as glutathione and dithiothreitol, lengthened the biotrophic stage and delayed symptom development, whereas an antioxidant, ascorbate, enhanced infection and promoted symptom development.

Engineered resistance to tomato spotted wilt virus in transgenic peanut expressing the viral nucleocapsid gene

The nucleocapsid gene of tomato spotted wilt virus Hawaiian L isolate in a sense orientation, and the GUS and NPTII marker genes, were introduced into peanut (Arachis hypogaea cv. New Mexico Valencia A) using Agrobacterium-mediated transformation. Modifications to a previously defined transformation protocol reduced the time required for production of transformed peanut plants. Transgenes were stably integrated into the peanut genome and transmitted to progeny. RNA expression and production of nucleocapsid protein in transgenic peanut were observed. Progeny of transgenic peanut plants expressing the nucleocapsid gene showed a 10- to 15-day delay in symptom development after mechanical inoculations with the donor isolate of tomato spotted wilt virus. All transgenic plants were protected from systemic tomato spotted wilt virus infection. Inoculated non-transformed control plants and plants transformed with a gene cassette not containing the nucleocapsid gene became systemically infected and displayed typical tomato spotted wilt virus symptoms. These results demonstrate that protection against tomato spotted wilt virus can be achieved in transgenic peanut plants by expression of the sense RNA of the tomato spotted wilt virus nucleocapsid gene

Color Mulches Influence Yield and Insect Pest Populations in Tomatoes

Field studies were conducted for three seasons, Fall 1988 and Spring and Fall 1989, on the effect of six mulch colors: blue, orange, red, aluminum, yellow, and white (fall) or black (spring), on fruit yields and on insect vectors of Sunny' tomato (Lycopersicon esculentum Mill.). Plant growth and yields were inconsistent with mulch colors during the three seasons. In Fall 1988, in a once-over harvest, extra-large (≥ 70 mm diameter) and marketable fruit yields were higher (P ≤ 0.05) on blue than on the conventional white mulch. In Spring 1989, early marketable yields on red mulch were higher than on black mulch, and in Fall 1989, under high stress from tomato mottle virus (TMoV) transmitted by silverleaf whitefly [Bemisia argentifolii (Bellows and Perring)], seasonal yield of extra-large fruit was better on orange than white mulch. In Fall 1988 and 1989, fruit size and marketable yields were reduced on yellow mulch. Aphids (Aphididae), thrips (Thripidae), and whiteflies were counted monthly in traps placed on the mulched beds. Aphids were least numerous on the aluminum and yellow and most numerous on the blue mulch. Where differences occurred, the fewest thrips were captured on aluminum and the fewest whiteflies were captured on the yellow, aluminum and orange mulches. Although differences were not always significant, the fewest adult whiteflies also were observed on foliage of tomato plants grown on these latter three mulches. Later in the seasons, as plant foliage covered the mulch, differences in the number of insects captured were similar for all mulch colors. Low numbers of whiteflies on the orange and aluminum mulches early in Fall 1989 delayed virus symptom development and increased yields. Virus symptom development was not delayed and yields were low on the yellow mulch, in spite of the low number of whiteflies. When averaged over all mulch colors, extra-large and marketable fruit yields increased linearly with delayed symptom development. It is proposed that, under high insect stress, mulches should be selected for their effects on insects in addition to their effects on soil temperature and plant morphology.

Transgenic tobacco plants expressing a truncated form of the PAMV capsid protein (CP) gene show CP-mediated resistance to potato aucuba mosaic virus.

Four constructs of the potato aucuba mosaic virus (PAMV) coat protein (CP) gene were engineered for expression in tobacco plants: The full-length CP sequence (FL CP), two truncated forms--one at the N terminus (46 amino acid residues are deleted) (NT138), one in the conserved core portion (86 amino acids deleted) (CT258) of the gene--and an antisense RNA construct. These constructs were introduced into tobacco plants (Nicotiana tabacum) via Agrobacterium tumefaciens-mediated transformation. The plants transformed with the NT138 and FL CP constructs produced the mRNAs and proteins from the respective transgene. Transformants with the CT258 construct produced the transgenic mRNA, but the modified CP was not detected in the 20 different transformants tested. Transgenic R0 and R1 tobacco plants expressing the full-length, CT258, and the antisense constructs exhibited protection to PAMV infection and a delay in symptom development when inoculated with 0.1 and 0.5 microgram/ml of purified PAMV. Transgenic plants expressing the NT138 construct did not confer any detectable protection to PAMV infection. These results suggest that an engineered coat protein mediated resistance (CPMR) can be obtained from a CP gene truncated in its core region. The role of the N-terminal domain of the CP in the CPMR of PAMV and the implication of either the RNA or the protein in the protection is discussed.

Nucleotide Sequence of the 3'-Terminal Region of Bean Yellow Mosaic Virus RNA and Resistance to Viral Infection in Transgenic Nicotiana benthamiana Expressing Its Coat Protein Gene.

The sequence of 4, 306 nucleotides from the 3'-poly(A) tract of bean yellow mosaic virus (BYMV) RNA has been determined. This sequence contained a single, long open reading frame (ORF), that when compared with amino acid sequences of other potyviruses was expected to encode carboxyl terminus of CI, 6K, NIa, NIb and coat protein (CP). The CP-coding sequence was predicted to be 819 nucleotides in length, encoding a protein of 273 amino acids. To produce transgenic plants resistant to viral infections, the BYMV CP gene was fused to a high expression promoter cassette containing a duplicated enhancer sequence of cauliflower mosaic virus 35S promoter followed by the 5'-untranslated sequence of tobacco mosaic virus RNA. The chimeric gene was introduced into Nicotiana benthamiana by Agrobacterium-mediated gene transfer, and the expression of CP gene was verified by Western blot analysis. Self-fertilized R1 progeny expressing detectable levels of CP showed significant delay in symptom development, as well as an attenuation of the symptoms.

Disease Resistance in Tobacco and Tomato Plants Transformed with the Tomato Spotted Wilt Virus Nucleocapsid Gene

The nucleocapsid protein (N) gene was cloned from the tomato spotted wilt virus (TSWV) Hawaiian L isolate. The 777-nucleotide N gene had 97 and 99% homology with a tomato (CNPH1) and two lettuce (L3 and BL) isolates, respectively. Leaf disks of Nicotiana tabacum cv. Xanthi nc and cotyledons of Lycopersicon esculentum cv. VF36 were transformed with Agrobacterium tumefaciens harboring a binary vector with a chimeric TSWV N gene cassette. Significantly lower numbers of local lesions and several days delay of symptom development were observed when R 1 transgenic tobacco plants expressing the TSWV N gene were challenged with TSWV. The R 1 transgenic tobacco plants transformed to produce an antisense RNA were as resistant as plants producing sense RNA [...]

Transgenic plants expressing a functional single-chain Fv antibody are specifically protected from virus attack.

Expression of viral genes in transgenic plants is a very effective tool for attenuating plant viral infection. Nevertheless, the lack of generality and risk issues related to the expression of viral genes in plants might limit the exploitation of this strategy. Expression in plants of antibodies against essential viral proteins could provide an alternative approach to engineer viral resistance. Recently, expression of complete or engineered antibodies has been successfully achieved in plants. The engineered single-chain Fv antibody scFv (refs 10, 11) is particularly suitable for expression in plants because of its small size and the lack of assembly requirements. Here we present evidence that constitutive expression in transgenic plants of a scFv antibody, directed against the plant icosahedral tombusvirus artichoke mottled crinkle virus, causes reduction of infection incidence and delay in symptom development.

The effect of elevated CO2 levels on the symptom expression of Colletotrichum gloeosporioides on avocado fruits

Exposure of freshly harvested avocado fruits to different concentrations of CO2 (11, 16 and 30%) for different lengths of time (4, 17 and 26 h) affected the decay development caused by Colletotrichum gloeosporioides. The delay in symptom development depended on the treatment given, the temperature regime of the fruit and time after harvest for treatment application. The most appropriate treatment was the application of 30% CO2 for 24 h at a temperature of 20‐25 C on the day of harvest. Treatment for shorter time periods, at lower temperatures or 50 h after harvest, resulted in a reduced response and, in some cases, enhanced symptom expression. Concentrations of 11 or 16% CO2 were less effective than 30% CO2 as the fruits became more mature. It is concluded that treatment of avocado fruits with high levels of CO: for a short period has the potential to provide an alternative means of controlling anthracnose.

Comparison of treatment strategies for Space Motion Sickness

Treatment strategies for Space Motion Sickness (SMS) were compared using the results of postflight oral debriefings. Standardized questionnaires were administered to all crewmembers immediately following Space Shuttle flights by NASA flight surgeons. Cases of SMS were graded as mild, moderate, or severe based on published criteria, and medication effectiveness was judged based on subjective reports of symptom relief. Since October 1989, medication effectiveness is reported inflight through Private Medical Conferences with the crew. A symptom matrix was analyzed for 19 crewmembers treated with oral combination of scopolamine and dextroamphetamine (scopdex) and 15 crewmembers treated with promethazine delivered by intramuscular i.m. or suppository routes. Scopdex has been given preflight as prophylaxis for SMS, but analysis showed delayed symptom presentation in 9 crewmembers or failed to prevent symptoms in 7. Only 3 crewmembers who took scopdex had no symptoms inflight. Fourteen out of 15 crewmembers treated with i.m. promethazine and 6 of 8 treated with promethazine suppositories after symptom development had immediate (within 1–2h) symptom relief and required no additional medication. There were no cases of delayed symptom presentation in the crewmembers treated with promethazine. This response is in contrast to untreated crewmembers who typically have slow symptom resolution over 72–96h. We conclude that promethazine is an effective treatment of SMS symptoms inflight. NASA policy currently recommends treating crewmembers with SMS after symptom development, and no longer recommends prophylaxis with scopdex due to delayed symptom development and apparent variable absorption of oral medications during early flight days.

Genetic engineering of potyvirus resistance using constructs derived from the zucchini yellow mosaic virus coat protein gene.

Three versions of the zucchini yellow mosaic virus (ZYMV) coat protein gene were engineered for expression in plants: the full-length coat protein sequence, the conserved core portion of the gene, and an antisense version. These constructs were introduced into muskmelon (Cucumis melo) and tobacco plants (Nicotiana tabacum) via Agrobacterium tumefaciens-mediated transformation; gene expression was verified by Northern and Western analysis. Transgenic R0 and R1 muskmelon plants expressing the full-length coat protein gene exhibited apparent immunity to ZYMV infection: There was a lack of symptom development during a 3-mo observation period and no measurable virus accumulation as determined by ELISA. Melon plants expressing the core or antisense constructs showed a several-day delay of systemic symptom development and reduction in virus titer. Furthermore, transgenic R1 tobacco plants expressing the full-length coat protein, core, or antisense constructs of ZYMV, a nonpathogen of tobacco, showed a short delay in symptom development and reduced virus titer when inoculated with the heterologous potyviruses, potato virus Y, and tobacco etch virus. The transgenic tobacco plants were not protected against the non-potyvirus, tobacco mosaic virus.

Modulation of natural resistance of avocado fruits to Colletotrichum gloeosporioides by CO 2 treatment

The level of the antifungal compound, 1-acetoxy-2-hydroxy-4-oxo-heneicosa-12,15-diene, in the peel of avocado fruits decreased during ripening. Fruits exposed to a high CO 2 concentration showed an initial increase in the diene level immediately after removal from CO 2. The concentration of the diene then significantly decreased, before a second increase in concentration. Exposure of avocado fruits to different concentrations of CO 2 (11, 16 and 30%) for different lengths of time (3, 10 and 24 h), enhanced the initial increase in diene concentration. Only 30% CO 2 applied for 24 h enhanced a significant second increase in diene level and delayed symptom development caused by Colletotrichum gloeosporiodes. Fruit exposure to lower CO 2 concentrations (16 and 11%) or short periods (3 and 10 h) caused no significant second increase and no significant effect on symptom development. Fruits exposed for 24 h to 30% CO 2 enhanced the concentration of the phenol epicatechin in a double-peak pattern similar to that exhibited by the diene. It is suggested that CO 2 treatments induce the enhancement of diene levels and consequently cause a delay in symptom development.

Protection against detrimental effects of potyvirus infection in transgenic tobacco plants expressing the papaya ringspot virus coat protein gene.

We obtained transgenic tobacco plants expressing the papaya ringspot virus (PRV) coat protein (CP) gene by transformation via Agrobacterium tumefaciens. Expression was effectively monitored by enzyme-linked immunosorbent assays (ELISA) of crude tissue extracts. Subcloned plants derived from eight original Ro transformants were inoculated with potyviruses: tobacco etch (TEV), potato virus Y (PVY), and pepper mottle (PeMV). Plants that accumulated detectable levels of the PRV CP showed significant delay in symptom development and the symptoms were attenuated. Similar results were obtained with inoculated R1 plants. We conclude that the expression of the PRV CP-gene imparts protection against infection by a broad spectrum of potyviruses.

Induction of a Symptom-Delaying Component in Tobacco Plants Infected with Cucumber Mosaic Virus.

Induction of a symptom-delaying component (SDC) in tobacco plants infected with cucumber mosaic virus (CMV) was investigated. Two largest leaves of Nicotiana tabacum cv. Xanthi seedlings at the 6-8-leaf stage were inoculated with a non-satellite-RNA-containing isolate of CMV-pepo at 100μg/ml and incubated for seven days at 25°C with a 16-hr photoperiod. With a sap-preparation from each leaf directly above the inoculated leaves (LAL) tobacco seedlings were treated and subsequently inoculated with CMV at 10μg/ml four days after the LAL-sap treatment. CMV symptoms were delayed in as many as 40% of the plants which had been treated with non-infectious LAL-sap, indicating that the LAL contained SDC. SDC was detected in the treated leaves at two to eight days after SDC-treatment and appeared to be translocated to the upper leaves at four to eight days in tobacco plants. SDC appeared to delay development of CMV symptoms in cucumber and in tomato. SDC also delayed symptom development of aphid-transmitted CMV in tobacco. In contrast, SDC did not affect local lesion formation on Chenopodium quinoa or Vigna unguiculata subsp. sesquipedalis by CMV nor symptom development in tobacco by tobacco mosaic virus or potato virus Y. In most of the tobacco plants repeatedly treated with SDC, symptom development of CMV was delayed for 4-20 days under field conditions. Antigenicity was detected intermittently in sap from some LAL by immunodiffusion tests using CMV antiserum. No CMV particles were observed in SDC-containing leaf sap. SDC was inactivated with a RNase A treatment. Gel electrophoresis showed that the corresponding bands with double-stranded RNAs 3 and 4 of CMV were detectable in. SDC-containing sap. However, treatment with single-stranded RNA 3 or RNA 4 failed to delay the development of CMV symptoms in tobacco plants.

The Genetic Engineering of Two Commercial Potato Cultivars for Resistance to Potato Virus X

We efficiently transformed the commercial cultivars of potato (Solanum tuberosum) Bintje, Desiree and Escort after optimizing the conditions for regeneration from potato tuber discs. For transformation, tuber discs were cocultivated with Agrobacterium tumefaciens using a disarmed binary vector system. This system allowed the introduction of a chimaeric gene encoding the coat protein (CP) of potato virus X (PVX) into two cultivars most susceptible to this virus, Bintje and Escort. Five transgenic plant lines with expression levels of CP higher than 0.1% of soluble leaf protein were analyzed for resistance to a challenging inoculation with PVX (1 μg/ml). We observed a delay in symptom development as well as a drastic reduction in the accumulation of virus. Furthermore, we found a correlation between the expression level of the CP–gene and the reduction in virus accumulation. Cytogenetic analysis of 62 independently obtained transgenic lines showed the normal tetraploid number of chromosomes (2n = 4x = 48) in 97 percent of the examined plants. Phenotypically all these plants appeared normal. One plant line exhibited an abnormal phenotype and contained about the octaploid number of chromosomes (4n ≈ 8x = 96). These results and preliminary data on morphological characteristics, as these are determined in the official variety registration procedure, indicate that potato cultivars can be genetically engineered to contain new desirable traits with preservation of their intrinsic properties.

Potential for biological control of Monilinia laxa in peach twigs

The antagonistic effect of four fungi ( Aspergillus flavus, Epicoccum nigrum, Penicillium frequentans and Penicillium purpurogenum) against Monilinia laxa on peach twigs was assayed in two field experiments in central Spain in spring and autumn. Results were different in the two periods. In spring, E. nigrum, P. purpurogenum and P. frequentans significantly reduced the success of infection when introduced before inoculation with the pathogen but A. flavus caused only a transient delay in the development of symptoms. However, when inoculation preceded antagonist introduction, only the treatments with P. frequentans resulted in a lasting reduction of symptoms and those with P. purpurogenum merely delayed symptom development. In autumn, only the treatments with E. nigrum resulted in a reduction of M. laxa infection. The potential for practical use of these fungi is discussed.

Inhibitors of avocado lipoxygenase: their possible relationship with the latency of Colletotrichum gloeosporioides

Linoleate oxidation by avocado lipoxygenase was inhibited in vitro in the presence of the specific inhibitor 5,8,11,14-eicosatetraynoic acid (ETYA). Infiltration with ETYA of avocado discs inoculated with Colletotrichum gloeosporioides delayed symptom development at concentrations where the fungus itself was not affected. Subsequently, a natural inhibitor of avocado lipoxygenase was isolated from peels of unripe avocado fruits and identified as epicatechin. It inhibited avocado lipoxygenase with a K i of 0·64 μm. The concentration of epicatechin in unripe fruits was 514 μg g − fresh weight of peel; this decreased during ripening to 8 μg g −1 fresh weight, before symptoms of C. gloeosporioides infections were expressed. A comparison of two cultivars with differing susceptibility to C. gloeosporioides showed that the concentration of epicatechin decreased faster in the cultivar in which symptoms appeared first. An atmosphere containing 50 μg l − ethylene enhanced the decrease of the lipoyxygenase inhibitor in avocado fruits and shortened the period before symptoms of disease were expressed. In over-mature, firm and naturally infected fruits hanging in the orchard the concentration of epicatechin was 260 μg g − in the area of the peel without symptoms and only 27 μg g − in that showing symptoms of infection. The results are discussed in relation to the hypothesis that the latency of the infection of avocado fruit by C. gloeosporioides may be accounted for by the degradation of the preformed antifungal compound, cis,cis-1-acetoxy-2-hydroxy-4-oxo-heneicosa-12, 15-diene, which is catalysed by avocado lipoxygenase, and that the in vivo lipoxygenase activity may increase during ripening owing to the decline in the levels of its endogenous inhibitor epicatechin.

Mycoplasma-like bodies in plants infected with potato witches' broom disease and the response of plants to tetracycline treatment

The virus origin of a Czechoslovak isolate of potato witches' broom disease is discounted: electron micrographs of ultrathin section of phloem tissues from plants infected with potato witches' broom disease demonstratedMycoplasma-like bodies, spherical or elongated showing binary fission and fragmentation. The minute corpuscles have a diameter of about 50–60 nm, the largest bodies of 1000 nm. The width of elongated filamentous structures is about 200 nm, most oval bodies have a diameter of 250 nm. A weak tetracycline treatment of diseased plants causes a delay of symptom development; a strong dose of tetracycline (applied by means of the wick method into the stem) inhibits symptom appearance completely. Tetracycline produces a phytotoxic effect inhibiting the growth of tomato plants and causing (at higher concentrations) necrosis and death of these plants. There is a note in the paper dealing with the term “mycoplasma”. The word mycoplasma in the sense ofEriksson (1897) or ofMereschkowsky (1910) does not correspond to the genus nameMycoplasma Nowak (1929).