Infection Of Leaf Discs Research Articles

Resistance estimation to Phytophthora palmivora in cacao genotypes using artificial inoculation and natural infection in the field

Black pod disease (BPD) is a severe biotic disorder affecting cacao trees in tropical regions generating an estimated global production reduction of approximately 20 to 30%. Accordingly, this study aimed to investigate the correlation between two artificial inoculation methods for Phytophthora palmivora and their potential association with natural infection in cacao clones. Incidence (%) and severity in detached pods (average lesion diameter) and leaf discs (disease severity index) were evaluated. The inoculation in pods at 6 DAI (Days After Inoculation) indicated the highest lesion diameter values for clone CCN51 (9.83 cm); hence, it was categorized as the most susceptible. Conversely, clones IMC67 (5.30 cm) and PA46 (5.27 cm), with the lowest lesion diameter values, were classified as moderately susceptible. Similar outcomes were observed in the leaf disc infection test, corroborating the susceptibility categorization of all six clones at 10 DAI. The leaf disc infection method showed a significantly positive correlation with the detached pod infection method, highlighting the feasibility of employing leaf inoculation to classify clones based on their susceptibility to BPD. Significant differences in aggressiveness were established between the isolates from different Colombian cacao regions. These findings were consistently reflected in the field, where the CCN51 clone exhibited the highest susceptibility compared to TSH565 and ICS95. This research proposes using the leaf technique to assess the aggressivity of Phytophthora palmivora isolates in cacao trees in Colombia.

Biocontrol Activity of Three Pseudomonas in a Newly Assembled Collection of Phytophthora infestans Isolates.

Late blight caused by the oomycete Phytophthora infestans constitutes the greatest threat to potato production worldwide. Considering the increasing concerns regarding the emergence of novel fungicide-resistant genotypes and the general demand for reducing inputs of synthetic and copper-based fungicides, the need for alternative control methods is acute. Several bacterial antagonists have shown anti-Phytophthora effects during in vitro and greenhouse experiments. We report the effects of three Pseudomonas strains recovered from field-grown potatoes against a collection of P. infestans isolates assembled for this study. The collection comprised 19 P. infestans isolates of mating types A1 and A2 greatly varying in fungicide resistance and virulence profiles as deduced from leaf disc experiments on Black's differential set. The mycelial growth of all P. infestans isolates was fully inhibited when co-cultivated with the most active Pseudomonas strain (R47). Moreover, the isolates reacted differently to exposure to the less active Pseudomonas strains (S19 and R76). Leaf disc infection experiments with six selected P. infestans isolates showed that four of them, including highly virulent and fungicide-resistant ones, could be efficiently controlled by different potato-associated Pseudomonas strains.[Formula: see text] Copyright © 2019 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Trichome expression of iaaM transgene influences their development and elongation in tobacco

The iaaM gene encodes a monooxygenase, an enzyme that can catalyze the synthesis of IAA from tryptophan and is functional in plants. We cloned the iaaM into the T-DNA region of a Ti binary vector pWM101 under the control of the cotton fiber specific E6 promoter, and the recombinant was transformed into Nicotiana tabacum WS38 via leaf disc infection with Agrobacterium tumefaciencs GV3101 that harbored the Ti plasmid. Some 25 transformed seedlings were screened out, and the trichomes of the transgenic leaves were both denser and lengthier. The E6 promoter-specific expression of iaaM in trichomes led to the more active auxin synthesis in the cells and allowed the transgenic leaves to develop tidier and longer trichomes. As more trichomes developed on the transgenic leaves, the leaf epidermis appeared fuzzier than the wild control. The research showed that iaaM expression in trichomes influenced the development of trichomes both during their initiation and elongation.

Climate favourability to leaf rust in Conilon coffee

The Brazil is the second largest producer of Conilon coffee (Coffea canephora), being the state of Espirito Santo responsible for producing 9.7 million bags of Conilon coffee. The biology of Hemileia vastatrix, etiologic agent of leaf rust, the main disease of this culture, is not well understood under the environmental conditions of Espirito Santo, Brazil. This study determined the ideal temperature and leaf wetness ranges for in vitro germination and infection of leaf discs in this pathosystem. For this, regression analyses with differents temperature and leaf wetness ranges were performed. The results showed that a climate characterized by mild mean temperatures (between 21.6 °C and 23.6 °C) with foliar wetness associated with high relative humidity (>80 %) is the most favorable condition for the infection of Conilon coffee by H. vastatrix. The knowledge of the optimal temperature and moisture conditions for the infection can also be useful for developing systems to predict the occurrence of the disease in C. canephora from Brazil.

Study on the transgenic tobacco expressing truncated wheat cold shock protein gene <I>TA3-13</I> and analysis of disease resistance

TA3-13 is a truncated gene coding for the fragment of wheat (Triticum aestivum L.) cold shock protein WCP1. It has been shown previously that the procaryotically expressed TA3-13 can induce resistance to Tobacco mosaic virus (TMV) when sprayed onto plant leaves. In this study, we constructed an expression vector pB-3-13 by cloning TA3-13 into the bionary vector pBI121 and transformed it into Agrobacterium tumefaciense strain EHA105 via freeze-thaw method. Tobacco (Nicotiana tobacum cv. Xanthi nc.) transformation was performed using the leaf disc infection method. After screening on MS medium containing kanamycin and PCR analysis, 33 T0 plantlets were identified as transgenic. Seeds from twenty T0 plants were collected and planted as T1 lines. Two T1 lines were selected for further characterization. PCR and GUS staining analysis showed that TA3-13 was integrated into the T1 tobacco genome and expressed. When inoculated on leaves, the transgenic tobacco showed significant resistance against TMV and rot pathogen Pectobactrium carotovorum subsp. carotovorum. These results suggest that the expression of TA3-13 in tobacco can induce defense responses to pathogen infection.

Producing IGF-1 in Transgenic Tobacco

In this research,a new expression vector of pCAMBIA1301-35S promoter-igf-1-nos,which included the gene of hIGF-1,was constructed.The gene is promoted by CaMV 35S promoter which can be expressed in tobacco in high performance.The expression vector of IGF-1 cDNA introduced by 35S promoter was transformed into LBA4404(Agrobacterium tumefacients),and then infected tobacco through leaf-disc infection.17 transgenic tobacco plants were gained after hygromycin selection,GUS test and PCR test of DNA.It shows IGF-1 gene can be transcribed into normal mRNA in transgenic tobacco by RT-PCR test.In this experiment,the expression of IGF-1 in tobacco lays the foundation for producing IGF-1 in tobacco and other dicotyledon bio-reactors as well.

Quantitative Trait Loci for Resistance to Powdery Mildew in Cucumber under Seedling Spray Inoculation and Leaf Disc Infection

AbstractPowdery mildew (PM) is an economically important disease of cucumber (Cucumis sativus L.) caused by the obligate biotrophic pathogen, Podosphaera xanthii, in greenhouse and field. In this study, a resistant breeding line S06 (greenhouse European type) and a susceptible cucumber inbred line S94 (Northern China open‐field type) and their F2:3 populations were used to investigate PM resistance under seedling spray inoculation in two growing seasons and leaf disc infection in one season. Quantitative trait loci (QTL) analysis was undertaken based on a constructed molecular linkage map of the corresponding F2 population using composite interval mapping. Five QTL (PM1.1, PM1.2, PM2.1, PM2.2 and PM5.1) for PM resistance were identified and located on linkage groups (LG) 1, 2 and 5, explaining 3.4–45.0% of the phenotypic variation. Three consistent QTL (PM1.1, PM2.1 and PM5.1) were detected under the three test conditions. The present results of the QTL analysis for PM resistance have laid a basis for breeding PM‐resistant cucumber varieties by molecular marker‐assisted selection.

The host guides morphogenesis and stomatal targeting in the grapevine pathogen Plasmopara viticola.

The oomycete grape downy mildew (Plasmopara viticola Berk. & Curt. Ex de Bary) is a serious pathogen of grapevine and spreads by extremely efficient cycles of asexual propagation. The high efficiency must involve efficient sensing of the host. We therefore analyzed the time course and morphology of the early development of this pathogen in a host system, by infection of leaf discs of grapevine (Vitis vinifera L. cv. Müller-Thurgau), and in a host-free system. Host factors were demonstrated to influence pathogen development in the following ways: (i) the release of zoospores from mature sporangia was accelerated, (ii) the morphogenesis of the germ tube was coordinated, and (iii) the zoospores were targeted to the stomata by factors that depended on stomata closure. The findings show that the early development of P. viticola is regulated, specifically and coordinately, by factors originating from the host plant.

Expression of immunogenic Puumala virus nucleocapsid protein in transgenic tobacco and potato plants.

Transgenic plants, expressing recombinant proteins, are suitable alternatives for the production of relevant immunogens. In the present study, the expression of Puumala virus nucleocapsid protein in tobacco and potato plants (Nicotiana tabacum and Solanum tuberosum) and its immunogenicity was investigated. After infection of leaf discs of SR1 tobacco and tuber discs of potato cv. "Desiree" with the Agrobacterium strain LBA4404 (pAL4404, pBinAR-PUU-S) containing the 1302 bp cDNA sequence of S-RNA segment of a Puumala virus, transgenic tobacco and potato plants expressed the Puumala virus nucleocapsid protein under control of the cauliflower 35S promoter. The recombinant proteins were found to be identical to the authentic Puumala virus nucleocapsid protein as analyzed by immunoblotting. Expression of the nucleocapsid protein was investigated over four plant generations (P to F4) and found to be stable (1 ng/3 microg dried leaf tissue). Transgenic tobacco plants were smaller compared to controls. The transformed potato plants were morphologically similar to control plants and produced tubers as the control potatoes. The S-antigen was expressed at a level of 1 ng protein/5 microg and 1 ng protein/4 microg dried leaf and root tissues, respectively, and remained stable in the first generation of vegetatively propagated potato plants. The immunogenicity of the Puumala virus nucleocapsid protein expressed in Nicotiana tabacum and Solanum tuberosum was investigated in New Zealand white rabbits. They were immunized with leaf extracts from transgenic tobacco and potato plants, and the serum recognized Puumala virus nucleocapsid protein. Transgenic plants expressing hantaviral proteins can thus be used for the development of cost-effective diagnostic systems and for alternative vaccination strategies.

Inhibition of lysine biosynthesis in Phytophthora infestans

Four inhibitors of the lysine biosynthetic enzyme dihydrodipicolinate synthase (DHDPS) were examined for their effects on the late blight fungus Phytophthora infestans . One of these compounds, dipicolinic acid, reduced mycelial growth of P. infestans by 61% and completely controlled blight infection of leaf discs when used at 1 mM. The other inhibitors, chelidamic acid, chelidonic acid and dimethyl cis -piperidine-2,6-dicarboxylate, were less effective against P. infestans in vitro and in vivo . The effects of dipicolinic acid and chelidonic acid on mycelial growth were partly overcome by the addition of lysine to the growth medium. Both of these compounds reduced DHDPS activity and lysine concentrations in P. infestans , although they had no effect on activities of aspartate kinase and diaminopimelic acid decarboxylase. Both dipicolinic acid and chelidonic acid reduced in vitro activity of DHDPS from P. infestans .

Resistance to Tomato Yellow Leaf Curl Geminivirus inNicotiana benthamianaPlants Transformed with a Truncated Viral C1 Gene

The C1 gene of tomato yellow leaf curl geminivirus (TYLCV) encodes a multifunctional protein (Rep) involved in replication. A truncated form of this gene, capable of expressing the N-terminal 210 amino acids (aa) of the Rep protein, was cloned under the control of the CaMV 35S promoter and introduced intoNicotiana benthamianausingAgrobacterium tumefaciens.The same sequence was also cloned in antisense orientation. When self-pollinated progeny of 19 primary transformants were tested for resistance to TYLCV by agroinoculation, some plants proved to be resistant, particularly in the sense lines. Two such lines were further studied. The presence of the transgene was verified and its expression was followed at intervals. All plants that were resistant to TYLCV at 4 weeks postinoculation (wpi) contained detectable amounts of transgenic mRNA and protein at the time of infection. Resistance was overcome in a few plants at 9 wpi, and in most at 15 wpi. Infection of leaf discs derived from transgenic plants showed that expression of the transgene correlated with a substantial reduction of viral DNA replication. Cotransfections of tobacco protoplasts demonstrated that inhibition of viral DNA replication requires expression of the truncated Rep protein and suggested that the small ORF C4, also present in our construct, plays no role in the resistance observed. The results obtained using both transient and stable gene expression systems show that the expression of the N-terminal 210 aa of the TYLCV Rep protein efficiently interferes with virus infection.

A putative IgG-binding 65 kDa adhesin involved in adhesion and infection of soybeans by Phytophthora megasperma f.sp. glycinea

Using an in vivo adhesion test and a newly developed leaf-disc infection assay we demonstrate that antibodies against mammalian substrate adhesion molecules, and also pre-immune IgG and D-mannose, inhibit (1) adhesion of the pathogen Phytophthora megasperma f.sp. glycinea (Kuan and Erwin) to the walls of soybean mesophyll cells, and (2) infection and colonization of the host through open wounds. Concanavalin A inhibited adhesion and infection in the infection assay but not adhesion in the adhesion test described previously. In control experiments, these compounds had no significant influence on cyst germination rates, or on germ tube growth of the pathogen. d -glucose was able to partially reverse the inhibitory effect of ConA and IgG. Intercellular washing fluids of soybean leaf tissue and surface (glyco)proteins from the pathogen were screened for putative IgG-binding adhesins. From both sources a major 65 kDa IgG-binding band was localized on SDS-PAGE gels. Based on ConA and IgG binding, the putative adhesins are (manno) glycoproteins. The study shows that adhesion of the fungal pathogen to the host cell wall is a prerequisite for colonization of the host tissue and that an IgG-binding 65 kDa glycoprotein appears to be instrumental in adhesion.

Insertional mutagenesis in Arabidopsis thaliana: isolation of a T-DNA-linked mutation that alters leaf morphology

We investigated the potential of the Agrobacterium tumefaciens T-DNA as an insertional mutagen in Arabidopsis thaliana. Arabidopsis lines transformed with different T-DNA vectors were generated using a leaf disc infection procedure adapted for efficient selection on either kanamycin or hygromycin medium. A standardized screening procedure was developed for the detection of recessive mutations in T2 populations of regenerated and/or transformed lines. Recessive mutations originating from the tissue culture procedure occurred at a low frequency - between 2% and 5%. Within 110 transformed lines that contained a total of about 150 T-DNA inserts, one recessive mutation, named pfl, cosegregated with a specific T-DNA copy. This pfl mutation mainly affected the morphology of the first seedling leaves under normal growth conditions and was mapped to chromosome 1. No recombination between the pfl locus and the kanamycin resistance marker on the T-DNA was detected when screening F2 and F3 populations of a mutant crossed to the wild type. The maximal genetic distance between the pfl locus and the kanamycin resistance gene, determined as 0.4±0.4 cMorgan, strongly suggests that the pfl mutation is induced by the insertion of the T-DNA. Our finding of one T-DNA-linked recessive mutation in 110 transgenic lines indicates that T-DNA can be used for mutagenization of the Arabidopsis genome under tissue culture conditions.

Effects of hornonal balance during leaf-disc infection of Nicotiana plumbaginifolia with Agrobacterium tumefaciens nopaline strain C58

Abstract The technique of leaf-disc transformation has been used in order to study the influence of different hormonal balances on the process of transformation of Nicotiana plumbaginifolia tissues with Agrobacterium tumefaciens nopaline-strain C58. C58 Agrobacterium strain incites withish, compact undifferentiated calli on Nicotiana tabacum, whilst allows the recover of shoots in N. plumbaginifolia. Shooting can be modulated through the hormonal balance of the culture medium mostly during the tissue culture phase (phase 2) subsequent to the period of infection (phase 1). Complete absence of hormones in phase 1 and 2 does not result in any callus formation or shooting on the edges of the infected leaf-discs, thus indicating that in this case the transformed cells need hormones at least in initial phase of development. In both the phases, addition of 2,4-D, NAA and kinetin resulted necessary in the stimulation of callus and shooting, namely in phase 2, and kinetin played a determinant role. Direct shooting...

The significance of temperature during sporulation on the biology of pycnidiospores of Mycosphaerella ligiilicola

SUMMARYThe germination, infectivity and survival of pycnidiospores obtained from cultures of Mycosphaerella ligulicola grown at 15 and 26 °C were compared. Spores formed at 26° (‘26° spores’) were less able to germinate at low relative humidities and showed a narrower temperature range for maximum germination after 6 h. At high spore densities 26° spores showed self‐inhibition of germination and, over a range of lower densities, growth of their germ tubes was checked, which resulted in lower infection of leaf discs compared with 15° spores in which this phenomenon did not occur.The fungus could be recovered from un‐sterile compost over a longer period after inoculation with 15° spores. Only after storage at a temperature well below zero was there a difference in viability between 15° and 26° spores.It is thought that the potential advantage of producing larger numbers of spores at 26° would be realized only under optimum conditions for dispersal and infection. The smaller number of spores produced at 15° are likely to be successful under natural conditions.