Leaf Assay Research Articles

SURVEILLANCE OF CITRUS CANKER IN PESHAWAR DIVISION AND ITS MANAGEMENT THROUGH HOST RESISTANCE

The present study was conducted to record incidence and severity of citrus canker and to screen citrus species against this disease. A detailed survey was conducted in 19 nurseries and 13 commercial orchards of Peshawar division during 2011. It was observed that almost all the nurseries were severely affected by the disease. The highest disease incidence (100%) and severity (31.54%) were observed in New Tarnab nursery in (Mention name of district here). Among the orchards, Shado II showed comparatively more disease severity (2.92%) as compared to other orchards. Bacteria, isolated from symptomatic plant samples on nutrient agar medium produced small, shiny yellow colonies with smooth margins. Biochemical tests further confirmed the bacteria as Xanthomonas axonopodis pv. citri. Detached and attached leaf assays were made to check the resistance of four citrus species i.e. Citrus sinensis (sweet orange), C. aurantifola (kaghazi lemon), C. paradise (Grape fruit) and C. medica (Lime). C. medica was moderately resistant whereas C. sinensis was found to be the most susceptible against the disease. Present study will be useful for developing best possible solution for the management of citrus canker in Peshawar division.

Techniques for screening Cercospora leaf spot resistant fenugreek genotypes

Cercospora leaf spot (CLS) caused by Cercospora traversiana is an important phyto-pathological problem of fenugreek (Trigonella foenum-graecum L.), a multiuse legume crop. Field screenings for resistant plants, although accurate and effective, demand significant time and a sizable workforce to accomplish. Also, weather conditions in the field may not always be favourable for uniform disease spread, which eventually may lead to failure of the overall experiment. Whole-plant assays (WPA) and detached leaf assays (DLA) with artificial inoculation not only help in scaling up the number of plants screened but also reduce the space, time, and amount of inoculum needed for the experiment. The results from our two experiments indicate that both the WPA and DLA methods can be used reliably to differentiate resistant and susceptible genotypes of fenugreek. In addition, the correlation coefficient between WPA and DLA (r = 0.875, P < 0.01), derived from the mean disease score of each genotype, shows that they can be used interchangeably while screening fenugreek for CLS. DLA was found to be temperature-sensitive for the development of CLS symptoms and wounded leaves developed symptoms faster than non-wounded leaves. These indoor methods can be used for the development of CLS-resistant fenugreek cultivars in areas where disease development is difficult under field conditions.

Detached Leaf Assays to Simplify Gene Expression Studies in Potato During Infestation by Chewing Insect Manduca sexta.

The multitrophic nature of gene expression studies of insect herbivory demands large numbers of biological replicates, creating the need for simpler, more streamlined herbivory protocols. Perturbations of chewing insects are usually studied in whole plant systems. While this whole organism strategy is popular, it is not necessary if similar observations can be replicated in a single detached leaf. The assumption is that basic elements required for signal transduction are present within the leaf itself. In the case of early events in signal transduction, cells need only to receive the signal from the perturbation and transmit that signal to neighboring cells which are assayed for gene expression. The proposed method simply changes the timing of the detachment. In whole plant experiments, larvae are confined to a single leaf which is eventually detached from the plant and assayed for gene expression. If the order of excision is reversed, from last in whole plant studies, to first in the detached study, the feeding experiment is simplified. Solanum tuberosum var. Kennebec is propagated by nodal transfer in a simple tissue culture medium and transferred to soil for further growth if desired. Leaves are excised from the parent plant and relocated to Petri dishes where the feeding assay is conducted with the larval stages of M. sexta. Damaged leaf tissue is assayed for the expression of relatively early events in signal transduction. Gene expression analysis identified infestation-specific Cys2-His2 (C2H2) transcription factors, confirming the success of using detached leaves in early response studies. The method is easier to perform than whole plant infestations and uses less space.

Detached Leaf Assays to Simplify Gene Expression Studies in Potato During Infestation by Chewing Insect Manduca sexta

Detached Leaf Assays to Simplify Gene Expression Studies in Potato During Infestation by Chewing Insect Manduca sexta

Difficulties in identifying Xanthomonas citri subsp. citri A pathotypes

A total of 350 citrus canker bacterial isolates, representing different citrus growing regions in Saudi Arabia, were identified as Xanthomonas citri subsp. citri (Xcc). DNA alignment of the 16S-rDNA of the three citrus canker organisms (Xcc, pathotypes A, X. fuscans pv. aurantifolii, pathotype B, and C) showed that they can be distinguished by using two SNPs. All the 350 Saudi Xcc strains belonged to the 16-rDNA haplotype of Xcc-A pathotypes. Pathogenicity in leaf assays using differential citrus cultivars, grapefruit (Citrus paradisi) and Mexican lime (C. aurantifolia), divided the Saudi Arabian bacterial isolates into two groups. The first group produced typical symptoms of Xcc, including erumpent canker lesions with water-soaked margins on all hosts. However, the second group caused atypical symptoms on grapefruit leaves. REP-PCR analysis grouped Xcc strains into three clusters that were not correlated with host, pathogenicity or geographic origin. However, the Baha Xcc population had the highest genetic variability, suggesting that this region may be responsible for disseminating Xcc strains to Saudi southern citrus-growing regions through movement of contaminated planting materials. Although we were able to definitely identify the Saudi citrus canker bacteria as Xcc-A pathotypes, its pathotypes still needs more investigation to be recognized. To our knowledge, this also is the first report of occurrence of citrus canker of sweet orange in the Abha region of Saudi Arabia.

Detached Leaf Assays for Resistance to Crown Rust Reveal Diversity Within Populations of Avena sterilis.

Crown rust is the most widespread and damaging disease of oat (Avena species). Genetic resistance to the pathogen is the preferred method for crop protection but widespread deployment of limited numbers of major effect genes has promoted the rapid emergence and spread of pathogen races that are able to overcome these genes. Combining genes with even partial resistance may help develop durable cultivars that are less vulnerable to changes in pathogen virulence. Partial resistance is expected to be relatively common in populations of wild species where constant pathogen pressure encourages diversity in host resistance mechanisms, but it may be discarded in conventional screens for major gene resistance. Here, we used a detached leaf assay to detect resistance to the crown rust pathogen, Puccinia coronata Cda. f. sp. avenae, in previously uncharacterized collections of the hexaploid wild oat relative A. sterilis made by the Polish National Centre for Plant Genetic Resources. Many of the accessions were collected in Morocco, the center of diversity for the Avena genus. The detached leaf assessment allowed individual plants to be challenged with multiple pathotypes and their responses compared with 34 known differentials. Broad-spectrum resistance was identified within accession PL 51855, which behaved as a single major locus on crossing to three cultivars. The locus provided resistance to over 50 rust pathotypes, a greater range than seen for any of the known host resistance (Pc) genes. Strong resistance was identified in other accessions, and heterogeneity in response within accessions was common. Several accessions show multiple partial resistance responses that may be of value for developing durable resistance in cultivars. Because the sources of resistance in all but two differential lines were collected outside of Morocco, resistance in all accessions tested here are potentially novel. This study demonstrates that diversity within A. sterilis accessions collected in Morocco could be a very valuable source of resistance to crown rust, and it provides new germplasm for use in resistance breeding programs. Detached leaf assessment provides a valuable first step in the identification of promising candidates in complex gene bank accessions.

Quantifying the Effects of a G137S Substitution in the Cytochrome bc1 of Venturia effusa on Azoxystrobin Sensitivity Using a Detached Leaf Assay.

The quinone outside inhibitor (QoI) fungicides are known for their inherently high resistance risk owing to substitutions in amino acid residues 129, 137, or 143 of the cytochrome b gene of phytopathogens. In Venturia effusa, cause of pecan scab, an intron adjacent to position 143 likely reduces this risk; however, the effects of a recently discovered substitution at position 137 (G137S) are unknown. Traditional in vitro assays are not useful for determining sensitivity of isolates of V. effusa to the QoI fungicides, owing to the fungitoxic effects of required alternative oxidase inhibitors. A detached leaf assay was developed to quantify the sensitivity of 59 isolates to azoxystrobin: 45 wild-type isolates and 14 carrying G137S. Isolate EC50 values ranged from <0.0001 to 0.3047 µg/ml; EC50 values for wild-type isolates ranged from <0.0001 to 0.2007 µg/ml (median 0.0023 µg/ml), whereas EC50 values for G137S isolates ranged from 0.0033 to 0.3047 µg/ml (median 0.0178 µg/ml). The median EC50 value for G137S isolates was significantly greater than that of the wild-type isolates; however, there was overlap between the two groups. This is the first report of sensitivity of V. effusa isolates to a QoI fungicide and evidence of G137S as a potential mechanism of partial resistance. However, although a complete control failure is unlikely, the impact of this substitution on QoI efficacy in Georgia pecan orchards remains to be determined.

Resistance Identification of Tree Peony Cultivars of Different Flowering Time to Gray Mold Pathogen Botrytis cinerea

Tree peonies are valuable ornamental plants and are widely cultivated in China and many other countries. Gray mold caused by Botrytis cinerea is an increasingly severe disease in Luoyang of China and seriously affects the ornamental value of tree peonies both in the open air and in greenhouses. However, the resistance of different tree peony cultivars to B. cinerea remains unknown. In this study, 15 tree peony cultivars belonging to three different flowering times were evaluated for resistance to B. cinerea by detached leaf assay measure. Results showed that the resistance of early-flowering peonies was stronger than that of later flowering peonies. Moreover, the correlation between flowering time and resistance of tree peonies was extremely significant (P &lt; 0.01). The information obtained in this study can provide theoretical basis both for further exploring the resistance genes of tree peony to B. cinerea and for the prevention and controlling of the gray mold.

Antagonistic activity of a novel antifungalmycin N2 from Streptomyces sp. N2 and its biocontrol efficacy against Rhizoctonia solani.

Antifungalmycin N2 (3-methyl-3,5-amino-4-vinyl-2-pyrone, C6H7O2N) is a novel bioactive substance produced by Streptomyces sp. N2. In this present work, the antagonistic activity of antifungalmycin N2 and its biocontrol efficacy on Rhizoctonia solani were carried out to evaluate its potential as a biocontrol agent against fungal plant diseases. By using potato dextrose agar media for in vitro cultivation of phytopathogenic fungi, the results showed that antifungalmycin N2 not only displayed broad-spectrum antifungal activities against various plant pathogenic fungi, but also had a strong antagonism to the sclerotial germination of R. solani. In a detached leaf assay, it was found that antifungalmycin N2 could effectively protect the rice leaves form the infection of R. solani, resulting in a significantly reduced sheath blight severity on the surfaces of rice leaves. In the pot experiments, the results also revealed that significantly lower sheath blight infections occurred in the tissues of the treated rice plants, which further confirmed that antifungalmycin N2 had a favorable biocontrol efficacy on rice sheath blight. In conclusion, the above results indicated that the novel antifungalmycin N2 was one of promising biocontrol agents for plant disease control.

Greater aggressiveness in the 2_A1 lineage of Phytophthora infestans may partially explain its rapid displacement of the US‐1 lineage in east Africa

The displacement in east Africa of the US‐1 clonal lineage of Phytophthora infestans by 2_A1 clonal lineage has been very rapid. This study tested the hypothesis that dominance of 2_A1 could be due, at least in part, to the increased aggressiveness of 2_A1 over US‐1, using both a detached leaf assay (DLA) and a tuber slice assay. The assays were conducted in Uganda and Kenya but US‐1 was only assayed in Uganda, because isolates could not be moved across borders and no potato US‐1 isolates were available in Kenya. All isolates were collected from potato and compared on two potato cultivars (Kachpot‐1 and Sarpo Mira), with the 2_A1 isolates also tested on tomato cultivar Rio Grande. Additionally, a tuber slice assay was done to test whether the capacity to infect tubers differed between 2_A1 and US‐1. The aggressiveness of the isolates in the DLA varied significantly both within and among isolates classified according to clonal lineage and for type of host. The 2_A1 isolates were significantly more aggressive than US‐1 isolates on both potato varieties evaluated. There were no significant effects of clonal lineage or potato cultivar used in the tuber assay. No significant correlation between foliar and tuber pathogenicity was observed. The 2_A1 isolates were significantly more aggressive on potato than on tomato. An effect of location was also observed in the DLA, on both hosts. It can be concluded from this study that greater pathogenicity of 2_A1 is at least partly attributable to its increased aggressiveness on potato.

Diagnosis of Fungal and Bacterial Diseases Based on Symptom &amp; Sign

Plant disease diagnosis is a form of hypothesis testing, where the hypothesis is simply the identity of the disease, and a good diagnostician goes through multiple iterations of the scientific method (seeking evidence through testing that supports or refutes the hypothesis that s/he generates). Identification of affected plants is one of the first steps in diagnosing a plant disease. Diagnosis is one of the most important aspects of a plant pathologist's training. Both scientific and common names of the plant should be noted. Without proper identification of the disease and the disease-causing agent, disease control measures can be a waste of time and money and can lead to further plant losses. Fungi are small, generally microscopic, eukaryotic, usually filamentous, branched, spore-bearing organisms that lack chlorophyll. Bacteria are prokaryotes. These are generally single-celled microorganisms whose genetic material (DNA) is not bound by a membrane and therefore is not organized into a nucleus. Our general objective was to acquaintance with Plant pathology laboratory materials & make disease diagnosis from field up to laboratory based on symptom and signs from different plant samples. All most all necro-tropic and bio-tropic fungi are grown on PDA agar, but bio-tropic fungi and bacterial diseases cannot grow on PDA agar. The best preferable method to diagnosis bio-tropic fungi is using direct leaf assay and blotting method on petri-dish. For bacterial disease identification we use NA agar. From this laboratory work I had concluded that those fungal and bacterial diseases need their niche to grow and well identified. It is very difficult to identify plant diseases based on sign and symptom. It is very imperative to diagnosis plant disease by collaborating both conventional (using sign and symptom) and molecular methods. It needs further work to identify rust diseases using race analysis up to f. species level.

Screening Vitis Genotypes for Responses to Botrytis cinerea and Evaluation of Antioxidant Enzymes, Reactive Oxygen Species and Jasmonic Acid in Resistant and Susceptible Hosts.

Botrytis cinerea is a necrotrophic fungal phytopathogen with devastating effects on many Vitis genotypes. Here, a screening of 81 Vitis genotypes for leaf resistance to B. cinerea revealed two highly resistant (HR), twelve resistant (R), twenty-five susceptible (S) and forty-two highly susceptible (HS) genotypes. We focused on the HR genotype, ‘Zi Qiu’ (Vitis davidii), and the HS genotype ‘Riesling’ (V. vinifera), to elucidate mechanisms of host resistance and susceptibility against B. cinerea, using detached leaf assays. These involved a comparison of fungal growth, reactive oxygen species (ROS) responses, jasmonic acid (JA) levels, and changes in the anti-oxidative system between the two genotypes after inoculation with B. cinerea. Our results indicated that the high-level resistance of ‘Zi Qiu’ can be attributed to insignificant fungal development, low ROS production, timely elevation of anti-oxidative functions, and high JA levels. Moreover, severe fungal infection of ‘Riesling’ and sustained ROS production coincided with relatively unchanged anti-oxidative activity, as well as low JA levels. This study provides insights into B. cinerea infection in grape, which can be valuable for breeders by providing information for selecting suitable germplasm with enhanced disease resistance.

Enhanced resistance to sclerotinia stem rot in transgenic soybean that overexpresses a wheat oxalate oxidase.

Sclerotinia stem rot (SSR), caused by the oxalate-secreting necrotrophic fungal pathogen Sclerotinia sclerotiorum, is one of the devastating diseases that causes significant yield loss in soybean (Glycine max). Until now, effective control of the pathogen is greatly limited by a lack of strong resistance in available commercial soybean cultivars. In this study, transgenic soybean plants overexpressing an oxalic acid (OA)-degrading oxalate oxidase gene OXO from wheat were generated and evaluated for their resistance to S. sclerotiorum. Integration and expression of the transgene were confirmed by Southern and western blot analyses. As compared with non-transformed (NT) control plants, the transgenic lines with increased oxalate oxidase activity displayed significantly reduced lesion sizes, i.e., by 58.71-82.73% reduction of lesion length in a detached stem assay (T3 and T4 generations) and 76.67-82.0% reduction of lesion area in a detached leaf assay (T4 generation). The transgenic plants also showed increased tolerance to the externally applied OA (60mM) relative to the NT controls. Consecutive resistance evaluation further confirmed an enhanced and stable resistance to S. sclerotiorum in the T3 and T4 transgenic lines. Similarly, decreased OA content and increased hydrogen peroxide (H2O2) levels were also observed in the transgenic leaves after S. sclerotiorum inoculation. Quantitative real-time polymerase chain reaction analysis revealed that the expression level of OXO reached a peak at 1h and 4h after inoculation with S. sclerotiorum. In parallel, a significant up-regulation of the hypersensitive response-related genes GmNPR1-1, GmNPR1-2, GmSGT1, and GmRAR occurred, eventually induced by increased release of H2O2 at the infection sites. Interestingly, other defense-related genes such as salicylic acid-dependent genes (GmPR1, GmPR2, GmPR3, GmPR5, GmPR12 and GmPAL), and ethylene/jasmonic acid-dependent genes (GmAOS, GmPPO) also exhibited higher expression levels in the transgenic plants than in the NT controls. Our results demonstrated that overexpression of OXO enhances SSR resistance by degrading OA secreted by S. sclerotiorum and increasing H2O2 levels, and eliciting defense responses mediated by multiple signaling pathways.

Metabolic Pathway of Topramezone in Multiple-Resistant Waterhemp (Amaranthus tuberculatus) Differs From Naturally Tolerant Maize.

Waterhemp [Amaranthus tuberculatus (Moq.) Sauer] is a problematic dicot weed in maize, soybean, and cotton production in the United States. Waterhemp has evolved resistance to several commercial herbicides that inhibit the 4-hydroxyphenylpyruvate-dioxygenase (HPPD) enzyme in sensitive dicots, and research to date has shown that HPPD-inhibitor resistance is conferred by rapid oxidative metabolism of the parent compound in resistant populations. Mesotrione and tembotrione (both triketones) have been used exclusively to study HPPD-inhibitor resistance mechanisms in waterhemp and a related species, A. palmeri (S. Wats.), but the commercial HPPD inhibitor topramezone (a pyrazolone) has not been investigated from a mechanistic standpoint despite numerous reports of cross-resistance in the field and greenhouse. The first objective of our research was to determine if two multiple herbicide-resistant (MHR) waterhemp populations (named NEB and SIR) metabolize topramezone more rapidly than two HPPD inhibitor-sensitive waterhemp populations (named SEN and ACR). Our second objective was to determine if initial topramezone metabolite(s) detected in MHR waterhemp are qualitatively different than those formed in maize. An excised leaf assay and whole-plant study investigated initial rates of topramezone metabolism (<24 h) and identified topramezone metabolites at 48 hours after treatment (HAT), respectively, in the four waterhemp populations and maize. Results indicated both MHR waterhemp populations metabolized more topramezone than the sensitive (SEN) population at 6 HAT, while only the SIR population metabolized more topramezone than SEN at 24 HAT. Maize metabolized more topramezone than any waterhemp population at each time point examined. LC-MS analysis of topramezone metabolites at 48 HAT showed maize primarily formed desmethyl and benzoic acid metabolites, as expected based on published reports, whereas SIR formed two putative hydroxylated metabolites. Subsequent LC-MS/MS analyses identified both hydroxytopramezone metabolites in SIR as different hydroxylation products of the isoxazole ring, which were also present in maize 48 HAT but at very low levels. These results indicate that SIR initially metabolizes and detoxifies topramezone in a different manner than tolerant maize.

Evolution and functional characterization of pectate lyase PEL12, a member of a highly expanded Clonostachys rosea polysaccharide lyase 1 family

BackgroundPectin is one of the major and most complex plant cell wall components that needs to be overcome by microorganisms as part of their strategies for plant invasion or nutrition. Microbial pectinolytic enzymes therefore play a significant role for plant-associated microorganisms and for the decomposition and recycling of plant organic matter. Recently, comparative studies revealed significant gene copy number expansion of the polysaccharide lyase 1 (PL1) pectin/pectate lyase gene family in the Clonostachys rosea genome, while only low numbers were found in Trichoderma species. Both of these fungal genera are widely known for their ability to parasitize and kill other fungi (mycoparasitism) and certain species are thus used for biocontrol of plant pathogenic fungi.ResultsIn order to understand the role of the high number of pectin degrading enzymes in Clonostachys, we studied diversity and evolution of the PL1 gene family in C. rosea compared with other Sordariomycetes with varying nutritional life styles. Out of 17 members of C. rosea PL1, we could only detect two to be secreted at acidic pH. One of them, the pectate lyase pel12 gene was found to be strongly induced by pectin and, to a lower degree, by polygalacturonic acid. Heterologous expression of the PEL12 in a PL1-free background of T. reesei revealed direct enzymatic involvement of this protein in utilization of pectin at pH 5 without a requirement for Ca2+. The mutants showed increased utilization of pectin compounds, but did not increase biocontrol ability in detached leaf assay against the plant pathogen Botrytis cinerea compared to the wild type.ConclusionsIn this study, we aimed to gain insight into diversity and evolution of the PL1 gene family in C. rosea and other Sordariomycete species in relation to their nutritional modes. We show that C. rosea PL1 expansion does not correlate with its mycoparasitic nutritional mode and resembles those of strong plant pathogenic fungi. We further investigated regulation, specificity and function of the C. rosea PEL12 and show that this enzyme is directly involved in degradation of pectin and pectin-related compounds, but not in C. rosea biocontrol.

Genomic distribution of a novel Pyrenophora tritici-repentis ToxA insertion element.

The ToxA effector is a major virulence gene of Pyrenophora tritici-repentis (Ptr), a necrotrophic fungus and the causal agent of tan spot disease of wheat. ToxA and co-located genes are believed to be the result of a recent horizontally transferred highly conserved 14kb region a major pathogenic event for Ptr. Since this event, monitoring isolates for pathogenic changes has become important to help understand the underlying mechanisms in play. Here we examined ToxA in 100 Ptr isolates from Australia, Europe, North and South America and the Middle East, and uncovered in isolates from Denmark, Germany and New Zealand a new variation, a novel 166 bp insertion element (PtrHp1) which can form a perfectly matched 59 bp inverted repeat hairpin structure located downstream of the ToxA coding sequence in the 3’ UTR exon. A wider examination revealed PtrHp1 elements to be distributed throughout the genome. Analysis of genomes from Australia and North America had 50–112 perfect copies that often overlap other genes. The hairpin element appears to be unique to Ptr and the lack of ancient origins in other species suggests that PtrHp1 emerged after Ptr speciation. Furthermore, the ToxA UTR insertion site is identical for different isolates, which suggests a single insertion event occurred after the ToxA horizontal transfer. In vitro and in planta-detached leaf assays found that the PtrHp1 element insertion had no effect on ToxA expression. However, variation in the expression of ToxA was detected between the Ptr isolates from different demographic locations, which appears to be unrelated to the presence of the element. We envision that this discovery may contribute towards future understanding of the possible role of hairpin elements in Ptr.

Non-host specificity of Botryosphaeriaceae on macadamia and blueberry

The ability of species in the Botryosphaeriaceae to inhabit seemingly healthy plants, reside as endophytes yet result in serious disease under stressful environmental conditions is poorly understood, as is their ability to colonise many unrelated hosts often without causing disease. Although many species of Botryosphaeriaceae have a wide host range, there is dearth of information on pathogenic variation of isolates of the same species on different hosts. In this study we used 16 isolates belonging to two genera of Botryosphaeriaceae from seven host plant species, to test the hypothesis that there are significant differences in pathogenic variation, varietal preferences and cross-pathogenicity in Botryosphaeriaceae on two unrelated host crops (macadamia and blueberry). Using the detached leaf and stem inoculation assays, we observed significantly (P < 0.05) high levels of pathogenic variation among isolates of Neofusicoccum parvum and compared with three other Neofusicoccum spp. and the three Lasiodiplodia spp. Host specificity was not observed among the isolates, as all were pathogenic on both hosts, but the aggressiveness of the isolates varied on the hosts, regardless of the source of the isolates. Between the host plants, blueberry was significantly more susceptible, regardless of the isolates than macadamia. There were significant (P < 0.03) differences among the four macadamia cultivars, isolates and cultivar × isolate interaction. Similar results for pathogenicity, aggressiveness and pathogenic variations were obtained using both the detached leaf and stem inoculation assays. Implications of these findings for disease management in cropping systems are discussed.

Reinforcement of resistance of modern rose to black spot disease via hybridization with Rosa rugosa

Most of the modern rose cultivars are susceptible to the highly destructive rose black spot disease caused by Marssonina rosae. In order to clarify the effect of hybridization with diploid Rosa rugosa on the morphological performance and black spot disease resistance of modern roses, we created the hybrid R. hybrida ‘Porcelina’ × R. rugosa ‘Dahong Zizhi’ and analyzed the morphological diversity and genetic constitution of 142 progeny. The progeny showed a wide range of variation, with a coefficient of variation of all traits of 40% and a maximum internode length of 118.9%. Cluster analysis of morphological traits and principal coordinate analysis of simple sequence repeats indicated that most of the progeny clustered with the female parent, while only a few individuals grouped with the male parent. Detached leaf assays were used to evaluate the black spot disease resistance of 75 lines and the two parents. The mean leaf area with symptoms (LAS) of the tested leaves of ‘Porcelina’ was 72.0 ± 12.9%, classifying it as highly susceptible, while R. rugosa showed high resistance to black spot disease. The coefficient of variation in LAS of the progeny was 68.1%, which indicates that the broad-spectrum resistance of rose black spot disease is controlled by multiple genes. A total of 11 progeny had an LAS < 10% and were significantly more resistant than the female parent. The results showed that the introduction of diploid R. rugosa into modern rose through conventional hybridization is effective at reinforcing resistance to black spot disease.

Fine mapping Ruv2, a new rust resistance gene in cowpea (Vigna unguiculata), to a 193-kb region enriched with NBS-type genes.

A new rust resistance gene Ruv2 was fine-mapped in cowpea to a 193-kb region on chromosome 2, which harboured 23 predicted gene models enriched with NBS-type genes. ZN016 is a landrace vegetable cowpea highly resistant to rust. Two previous studies using mixed-spores inoculation suggested different modes of inheritance of rust resistance in ZN016. In this study, we initially developed a detached leaf assay with a purified single-rust isolate (Auv-LS). Using this approach, we assessed the inheritance of rust resistance in a recombinant inbred line (RIL) population and an F2 population, both derived from the cross of "ZN016" and the susceptible cultivar "Zhijiang282." A single dominant gene mode against Auv-LS was revealed in both populations. QTL mapping showed that this gene was coincident with the Ruv2 locus on LG7, one of the three resistance QTLs previously mapped based on mixed-spores inoculation data. Therefore, Ruv2 was considered as specifically against the rust isolate Auv-LS. Through an analysis of the RIL recombinants at Ruv2, we fine-mapped the gene to an ~ 0.45-cM interval between SNP markers 2_09656 and 2_00973, which corresponded to an ~ 193-kb region on chromosome 2 that harboured 23 predicted gene models enriched with NBS-type genes. Re-sequencing of the two parents revealed polymorphisms in four genes predictively to cause substantial protein structural changes, rendering them valuable candidate genes for future validation. Cross-species syntenic analysis indicated that Ruv2 may represent a novel rust resistance gene in food legumes. A cleaved amplified polymorphic sequences marker tightly linked to Ruv2 was developed to facilitate breeding. This work establishes a basis for map-based cloning of Ruv2 and breeding for rust resistance in cowpea and other legume crops.

Comparison of Whole Plant and Detached Leaf Screening Techniques for Identifying Anthracnose Resistance in Strawberry Plants.

Anthracnose is a destructive disease of strawberry caused by several Colletotrichum species including C. acutatum, C. fragariae, and C. gloeosporioides. Identification of anthracnose resistant strawberry germplasm has commonly relied on inoculation of whole plants with isolates of these pathogens. In this study, whole plants and detached leaves from 81 germplasm lines were inoculated with a conidial suspension of isolates of C. acutatum, C. fragariae, and C. gloeosporioides, incubated in the dark at 30°C, 100% relative humidity, for 48 h, and assessed for disease severity based on symptoms on inoculated petioles and leaves. The correlation between the disease severity ratings of the whole plants rated 30 days after inoculation and detached leaves rated 5 days after inoculation was determined. Based on leaf symptoms and petiole lesions, the association between the whole plant leaf disease severity rating (DSR) and detached leaf DSR was positive (rp = 0.70), and the association between the whole plant DSR and the detached leaf DSR was also positive (rp = 0.66). Whole plant and detached leaf DSRs were used to assign each germplasm line to a resistance category, and a posthoc Tukey's test showed that the whole plant DSR means and the detached leaf DSR means for each resistance category differed significantly at p < 0.05. This research was used to develop a strawberry detached leaf assay which can reliably and quickly determine the degree of resistance of strawberry germplasm to anthracnose.