Leaf Disc Samples Research Articles

Evaluation of CRISPR/Cas12a-based DNA detection for fast pathogen diagnosis and GMO test in rice

DNA test is broadly used in diagnosis of crop disease and identification of genetic modified organism (GMO) in agriculture. However, rapid, low-cost, user-friendly, and field-deployable DNA test method is still limit. Recently, the RNA programmable nuclease of CRISPR/Cas is engineered as a new nucleic acid detection platform, but their application in plant remains to explore. In this study, we evaluated the Cas12a-based DNA detection for crop disease diagnosis and GMO test. A total of 14 crRNAs were designed to target two Magnaporthe oryzae genes and a synthetic Cry1C gene which encodes Bacillus thuringiensis δ-endotoxin and has been used to develop transgenic rice cultivar (Bt-rice) in China. Using a fluorescent reporter, targeted genes were easily detected by LbCas12a after recombinase polymerase amplification (RPA) for all crRNAs, despite that the signal strength varied 2–3-folds between different crRNAs. We further combined the filter paper-based DNA extraction and lateral flow assay (LFA) with RPA-Cas12a for DNA detection. This optimized Cas12a diagnostics method is carried at body temperature and does not require extra instrument except filter paper and LFA strip. Our data show that rice blast pathogen and Bt-rice were efficiently identified from leaf disc samples using this optimized DNA test method with highly active crRNAs. Moreover, LbCas12a exhibited variable nuclease activities on different targets; therefore, highly active crRNA is critical for successful DNA test using Cas12a and LFA. Owing to its simplicity, efficiency, and low-cost, DNA test using CRISPR/Cas12a would be easily applied in field for crop disease diagnosis and GMO administration.

A High-Throughput Phenotyping System Using Machine Vision to Quantify Severity of Grapevine Powdery Mildew.

Powdery mildews present specific challenges to phenotyping systems that are based on imaging. Having previously developed low-throughput, quantitative microscopy approaches for phenotyping resistance to Erysiphe necator on thousands of grape leaf disk samples for genetic analysis, here we developed automated imaging and analysis methods for E. necator severity on leaf disks. By pairing a 46-megapixel CMOS sensor camera, a long-working distance lens providing 3.5× magnification, X-Y sample positioning, and Z-axis focusing movement, the system captured 78% of the area of a 1-cm diameter leaf disk in 3 to 10 focus-stacked images within 13.5 to 26 seconds. Each image pixel represented 1.44 μm2 of the leaf disk. A convolutional neural network (CNN) based on GoogLeNet determined the presence or absence of E. necator hyphae in approximately 800 subimages per leaf disk as an assessment of severity, with a training validation accuracy of 94.3%. For an independent image set the CNN was in agreement with human experts for 89.3% to 91.7% of subimages. This live-imaging approach was nondestructive, and a repeated measures time course of infection showed differentiation among susceptible, moderate, and resistant samples. Processing over one thousand samples per day with good accuracy, the system can assess host resistance, chemical or biological efficacy, or other phenotypic responses of grapevine to E. necator. In addition, new CNNs could be readily developed for phenotyping within diverse pathosystems or for diverse traits amenable to leaf disk assays.

First Report of QoI Insensitive Cercospora beticola on Sugar Beet in Ontario, Canada.

Cercospora beticola Sacc. causes Cercospora leaf spot (CLS) of sugar beet (Beta vulgaris L.) and is the most destructive foliar disease of sugar beet worldwide (1). The QoI fungicide pyraclostrobin has been an important management tool for CLS in Canada since 2003. Beginning in 2010, some growers reported poor disease control after applying pyraclostrobin. Leaf disk samples with CLS lesions were collected in September 2012 from 16 commercial fields located in Kent and Lambton Counties, Ontario, Canada. These counties (ca. 300,000 ha) encompass the major commercial sugar beet production area in Ontario (ca. 3,925 ha). CLS severity ranged from low to severe among the sampling sites. Leaf discs with a single leaf spot were cut from leaves using a hole punch. Spots were up to 5 mm in diameter with tan, light brown, or sometimes gray centers. DNA was extracted from leaf discs using a Qiagen DNeasy Plant Mini Kit (Germantown, MD) according to the manufacturer's instructions. PCR was used to amplify a fragment of the C. beticola cytochrome b (CYTB) gene (4). Pure cultures were obtained by placing plant tissue in a moist chamber and transferring single spores to V8 juice agar. PCR products were sequenced for 32 samples at the Genomics Technology Support Facility (Michigan State University, East Lansing, MI) and 25 were confirmed to have 100% identity with the sequence of QoI-resistant C. beticola from Michigan (2) and to QoI-resistant isolates from GenBank (Accession Nos. JQ619933 and JQ360628). The remaining seven had 100% identity with a sensitive isolate (EF176921.1). Each resistant isolate contained a change in codon 143 that is predicted to lead to a substitution of G143A in the cytochrome b gene. This G143A mutation has been associated with QoI resistance in a number of fungi (3). To confirm the result, a conidium germination bioassay was carried out using nine isolates with the G143A mutation on sugar beet leaf agar covered with water agar amended with pyraclostrobin at concentrations ranging from 0 to 54.3 μg/ml and distributed on a spiral gradient using an Eddyjet II spiral plater. The medium was supplemented with salicylhydroxamic acid (SHAM) to block the alternate oxidation pathway. Following incubation at 25°C for 2 days, the distance between the center of the plate at which conidial germination was 50% of the maximum observed growth (EC) and the point at which conidial germination terminated were measured (TEC). The EC50 values were determined from the SGE software for each isolate by entering the EC and TEC values, respectively. The estimated EC50 for a representative wild type (sensitive) isolate was 0.03 μg/ml, while the value for the resistant isolate could not be calculated because it was greater than the highest concentration tested (54.3 μg/ml). Additionally, in the controls with no SHAM or fungicide, the resistant isolate showed a consistent reduced germination rate compared to the sensitive isolate (30.0% and 93.5% germination, respectively). Confirmation of fungicide insensitivity will require a re-evaluation of current management practices in Ontario to minimize economic losses due to CLS.

Aphids secrete watery saliva into plant tissues from the onset of stylet penetration

Aphid feeding requires the secretion of two types of saliva: gelling saliva (from the principal gland) that forms an intercellular sheath for the penetrating stylet, and watery saliva [from accessory salivary glands (ASGs)] that facilitates intracellular penetration and phloem feeding. Plant viruses can be used as salivary markers to investigate key steps in aphid feeding, and penetration can be monitored electrically using the electrical penetration graph (EPG) approach. We conducted a series of EPG-controlled transmission experiments using Cucurbit aphid-borne yellows virus [CABYV; Polerovirus spec. (Luteoviridae)], which is retained in the ASGs, as a marker for watery saliva secretions. The melon aphid, Aphis gossypii Glover (Hemiptera: Aphididae), was used as a vector and melon seedlings, Cucumis melo L. (Cucurbitaceae), as host plants. Viruliferous aphids were interrupted at various stages during stylet penetration, i.e., during intercellular penetration prior to intracellular puncture and following a potential drop within the first probe. Viruliferous aphids and leaf disc samples obtained from the stylet penetration site were used to detect CABYV by quantitative real-time RT-PCR. Approximately half of the inoculated leaf discs were found to be infected with CABYV after very brief (12.9 ± 1.9 s) intercellular stylet probes and before intracellular stylet puncture. The number of virus particles ejected during such probes was similar to the number ejected by aphids during longer probes including a single intracellular puncture. Our results therefore suggest that watery saliva is secreted by aphids from the onset of stylet penetration.

Feasibility analysis of leaf disc samples produced via agroinfiltration for promoter trapping studies<br><br>

Promoter trapping is a method used to isolate and characterize regulatory regions from genomes by elucidating the expression of a promoterless reporter gene flanked by two transposable elements. The conventional method of promoter trapping requires the generation of stable transformed tissue culture derived plant lines. Nevertheless,this method can be laborious, time consuming and expensive. As an alternative method, leaf disc samples produced via leaf agroinfiltration was employed in this work. A promoter trapping construct named pCAMDIN was created which contains a promoterless GUS (?-glucuronidase) gene flanked by the left and right T-DNA border using pCAMBIA 1301 and pBI 121. Following that,the protocol for agroinfiltration of tomato plants using both direct agroinfiltration and vacuum agroinfiltration was optimized. Non-destructive protocols for detection of GUS genes were tested and optimised. Following that, GUS gene expression was studied and areas which showed expression were isolated by making leaf punches. Analysis was carried out by Southern blotting and T-DNA fingerprinting to determine the gene copy number.

Sulphur supply and infection with Pyrenopeziza brassicae influence L-cysteine desulphydrase activity in Brassica napus L.

Different field surveys have shown that sulphur (S) fertilization can increase the resistance of agricultural crops against fungal pathogens. The mechanisms of this sulphur-induced resistance (SIR) are, however, not yet known. Volatile S compounds are thought to play an important role because H(2)S is toxic to fungi. A field experiment was conducted to analyse the influence of S fertilization and the activity of H(2)S-releasing enzymes on fungal infections. Two levels of N and S fertilizers and two varieties of oilseed rape were investigated with respect to their potential to release H(2)S by the enzymatic activity of L-cysteine desulphydrase (LCD) and O-acetyl-L-serine(thiol)lyase (OAS-TL). LCD releases H(2)S during cysteine degradation, while OAS-TL consumes H(2)S during cysteine synthesis and free H(2)S is only released in a side reaction. All plots of the field trial showed an infection with Pyrenopeziza brassicae and leaf disc samples were taken from visibly infected leaf areas and apparently uninfected areas to investigate the reaction to the infection in relation to the treatments. Different S fractions and the activities of LCD and OAS-TL were measured to evaluate the potential to release H(2)S in relation to S nutrition and fungal infection. S fertilization significantly increased the contents of total S, sulphate, organic S, cysteine, and glutathione in the plants, but decreased LCD activity. Infection with P. brassicae increased cysteine and glutathione contents, as well as the activity of LCD. Therefore crops were able to react to a fungal infection with a greater potential to release H(2)S, which is reflected by an increasing LCD activity with fungal infection.

Field Resistance to Bemisia tabaci in Cucumis melo

The whitefly Bemisia tabaci (Gennadius) B-Biotype is a major pest on cucurbits in the Caribbean islands. Five field trials were conducted to identify resistance among 80 genotypes of Cucumis melo L. from diverse geographic origins. We focused on resistant rather than tolerant genotypes by counting adults on the abaxial side of two leaves of each plant at least three times in each trial, and larval density of under leaf disk samples at least twice in each trial. On the basis of insect density, three Indian accessions, PI 414723, PI 164723, and 90625, and one Korean accession, PI 161375, had field resistance. On those accessions, we observed 3.6 to 6 times fewer adults than on the most susceptible genotypes (`AR Top Mark' or `Délice de Table') and 11 to 29 times fewer larvae than on `AR Top Mark' or B66-5. Those levels of resistance may be sufficient to significantly reduce pesticide use in Guadeloupe (Lesser Antilles) where B. tabaci populations are lower than in the Southern United States or in the Mediterranean Basin. Higher levels of resistance are needed for genetic control, and may be achieved by a combination of different partial resistance genes.

Influence of Drought-Induced Water Stress on Soybean and Spinach Leaf Ascorbate-Dehydroascorbate level and Redox Status.

We examined the influence of water stress (water deficit) induced by drought on the steady state levels of ascorbic acid (ASC), dehydroascorbate (DHA), and the ASC&rcolon;DHA redox status in leaflets of Glycine max (soybean) and leaves of Spinacia oleracea (spinach). Two soybean cultivars (cv. Essex and cv. Forrest) and one spinach cultivar (cv. Nordic) were grown in high-light growth chambers ( approximately 1000-1200 µmol m-2 s-1) or in the greenhouse during May, June, and July 1999. The cultivars were supplied with water until approximately 25-29 d postemergence, at which time one-half of the plants were not watered for a period of from 4.5 to 7.5 d; the other half of the plants were provided water daily and served as controls. On designated days, leaf water potential (PsiLeaf) was measured, and leaf disks of constant area were excised in the period between approximately 1230 and 1330 hours. Leaf disk samples were immediately frozen in liquid N2, samples were extracted, and ASC and DHA levels were measured and expressed as µmol per gram dry mass per time point. For the soybean cultivars, low PsiLeaf values ( approximately -3.00 to -3.95 MPa) were accompanied by slight decreases in ASC levels and slight increases in DHA levels per gram dry mass. In some cases, leaflet ASC levels of water-stressed soybeans were similar to controls or were even increased by as much as 1.2 times. In soybeans, the mole fraction of ASC remained at 93-99 mol% of the total ascorbate (ASC+DHA), indicating that most of the total ascorbate remained in the reduced form even at low water potential. In spinach plants subjected to water stress (-1.8 to -2.6 MPa), leaf ASC decreased as much as 38%, but the ASC remained at 96-99 mol% of the total ascorbate. It is concluded that during water stress, enzymes of the ascorbate-glutathione cycle in leaf mesophyll cells, as well as in the system that generates reductant to support DHA to ASC recycling, e.g., photosynthetic electron transport in chloroplasts, is able to remain active enough to maintain reduction of DHA to ASC.

Survival and persistence of bioluminescent Xanthomonas campestris pv. campestris on host and non‐host plants in the field environment

Dispersal and persistence of a pathogenic strain of Xanthomonas campestris pv. campestris, genetically engineered to bioluminesce, was followed in and on host and non‐host plants in the field environment. Black rot susceptible cabbage plants were mist inoculated with the bioluminescent strain only, or were mist inoculated with X. campestris pv. vesicatoria or a weakly pathogenic strain of X. c. campestris 1 week before challenge inoculation with the bioluminescent strain. Growth of the bioluminescent strain was detected with a low‐light, charge‐coupled device camera or through bioluminescence measurements of broth‐enrichment cultures of leaf disk samples. Bioluminescent X. c. campestris could often be observed as populations on symptomless leaves or in lesions, and persisted as a vascular endophyte for more than 6 months throughout the winter growing season. Dispersal to cruciferous and non‐cruciferous weeds was frequently detected. Pre‐inoculation with X. c. vesicatoria or the weakly pathogenic X. c. campestris did not significantly affect the movement and persistence of the bioluminescent strain nor reduce the incidence of black rot disease.

Measurement of acephate and its principal metabolite in leaves by gas‐liquid chromatography

AbstractA semi‐micro method is described for monitoring levels of acephate (O, S‐dimethyl acetylphosphoramidothioate) and its principal metabolite, methamidophos (O, S‐dimethyl phosphoramidothioate) in leaf material derived from treated, hydroponically grown gerberas (Gerbera jamesonii H. Bollus, ex Hook. f.) To minimise injury to the living leaf, analyses were performed on 13‐mm diameter discs punched from the interveinal regions of the lamina. The leaf disc samples were then extracted with ethyl acetate. Clean‐up of extracts was accomplished by passing the filtered leaf extracts through glass mini‐columns containing carbon + filter aid (2 + 5 by mass). Acephate and methamidophos were eluted from the column with methanol and measured simultaneously by programmed temperature gas chromatography using a thermionic specific detector.

Effects of Ozone and Acid Rain on Pesticide Degradation, Delaware, CO., OH., 1986

Abstract Two-year-old red and white oak seedlings were sprayed with insecticides to determine if chemical efficacy against gypsy moth (GM) larvae was affected by exposure to various levels of atmospheric deposition. The experimental parameters (factorial design) were 2 insecticides (Baythroid, Dylox); 3 concentrations (V4, 1, 2 × recommended rate); 3 simulated acid rain (SAR) treatments (3.0 pH, 3.5 pH, 4.2 pH); 2 ozone (O3) exposures (0.15 ppm, ambient); and 3 replicates. Seedlings were sprayed to runoff on 30 Jun (22�C, 45% RH, 0 mph wind) using a 11.4-liter compressed CO2 driven R&D Model TS backpack sprayer equipped with a no. 5500 adjustable cone tip nozzle opened Vi turn from full closed at 2.1 kg/cm2 pressure. To establish initial chemical efficacy, 2 leaf disc samples (1.8 cm2 each were removed from leaves (24 h postspray) on each seedling for GM bioassay. Seedlings were then subjected to the appropriate SAR and O3 treatments weekly for 3 weeks. These treatments were 45 min SAR (1.27 cm) followed by fumigation for 3 days at 7 h day. At 22 days postspray, seedlings were resampled to determine degradation effects. For both bioassays, leaf disc samples were placed on moistened filter paper in plastic petri dishes. Two 2nd-instar GM larvae were added to each test dish and allowed to feed for 24 h. Average % mortality of larvae was used to measure effects.

A Conductivity Method for Screening Populations of Eucalypts for Frost Damage and Frost Tolerance

The assessment of frost tolerance of Eucalyptus leaf tissue by an electrical conductivity method is described. Leaf disc samples were exposed to freezing treatments in test tubes within a liquid cold bath with precise temperature control, and the conductivity of the disc leachate measured. Leaf discs were then frozen at -20°C to obtain an absolute conductivity for the tissue. A modified relative conductivity value (RC*) is presented together with calibration against visual observations of leaf damage for seedlings of E. regnans and E. delegatensis. RC* was found to be a reliable indicator of leaf survival status and the RC*-damage relationship was constant across provenances of both species. Provenance and family variation for frost tolerance were examined for both species and significant differences in tolerance levels were found for unhardened E. delegatensis and for hardened seedlings of both species at several test temperatures. Provenance rankings were identical to results from field trials reported in the literature. The effect of the period between frost treatment and conductivity measurement was assessed together with variation between leaves of the same plant and the effect of storage of cut leaves for 24 h prior to exposure to frosting treatments. The absence of any significant effects from storing leaves for 24 h indicates the potential of using the method to screen samples collected in the field. Problems of relating RC*, leaf damage and survival of the whole plant are discussed and the potential use of RC* as a simple screening technique for identifying frost-tolerant families and provenances is examined.

Persistence on cotton foliage of insecticide residues toxic to Heliothis larvae

AbstractControl measures for Heliothis spp. on cotton are usually applied against eggs and newly hatched larvae that infest plant terminal growth. In order to investigate the rate of degradation of a range of insecticides, leaf disc samples were collected, at intervals after application, from both fully expanded cotton leaves and unfolding terminal leaves. The samples were subjected separately to bioassay with newly hatched larvae, and to chemical analysis of the surface deposits.On old leaves, larval mortality caused by two biological insecticides declined by half after field exposure for 36 h, but for the synthetic insecticides, the interval ranged from 4.5 days with endosulfan to 18 days with fenvalerate. On new leaves, the longest interval was 7 days with fenvalerate. Chemical half‐life on old leaves ranged from 1 day with endosulfan to 12 days with DDT, and was also shorter on young leaves than the equivalent biological half‐life. Bioassay appears to be the most reliable method of assessing insecticidal activity over time. Dosage/mortality data were integrated to give LC50 and LC90 values.Leaf expansion may have the greatest effect on insecticide persistence during the 6 week period of rapid growth commencing 2 months after sowing. Expansion was slower when plants were water stressed, and accelerated following irrigation. The implications of the above findings for cotton pest management are discussed.

Radiosensitivity of Phaseolus vulgaris L. cv. blue lake: Morphological and physiological criteria

Mujeeb K. A. and Greig J. K. `Radiosensitivity of Phaseolus vulgaris L. cv. Blue Lake : morphological and physiological criteria. Radiation Botany 12, 437–439, 1972.-Phaseolus vulgaris L. cv. Blue Lake seeds were given acute gamma radiation exposures of 1·0, 1·5, 2·0, 2·5, 3·0, 3·5, 4·0, 4·5, 5·0 and 7·5 kR from a 4500 Ci 60Co source. Leaf-disk samples from primary leaves, collected 21 days after seeds were planted, were analyzed for total chlorophyll. Germination and seedling height, determined at the same time, were used to estimate seedling performance, (germination x seedling height, as per cent of respective controls). Leaf spotting progressed with ascending dosage. Highly significant negative correlations to exposure were found for total chlorophyll content and seedling performance. The morphological and pigment criteria differed significantly in estimating biological response, yielding D50 values of 4·5 kR and 6·3 kR, respectively. The pigment criterion has been suggested to provide greater mutability in M2.

A Rapid Method for Obtaining Leaf Samples for Electron Microscopy

Abstract This report, describes a rapid and convenient method to obtain leaf disc samples for electron microscopy. It was developed in connection with bio-chemical-ultrastructural studies of herbicidal effects on protein synthesis. It was important to speed up the sampling time and to obtain uniformly sized samples.

A Rapid Method for Obtaining Leaf Samples for Electron Microscopy

Abstract This report, describes a rapid and convenient method to obtain leaf disc samples for electron microscopy. It was developed in connection with bio-chemical-ultrastructural studies of herbicidal effects on protein synthesis. It was important to speed up the sampling time and to obtain uniformly sized samples.