Detached Leaf Assay Research Articles

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

Film-Forming Polymers and Surfactants Reduce Infection and Sporulation of Phytophthora ramorum on Rhododendron.

Phytophthora ramorum, cause of sudden oak death and ramorum leaf blight, can persist undetected in infested nurseries. Many conventional fungicides are effective in reducing or delaying symptom expression but some may confound visual detection of infected plants. We tested film-forming polymers (FFPs) and surfactants for their ability to reduce infection and sporulation of P. ramorum on rhododendron. FFPs (Anti-Stress, Moisturin, Nature Shield, Nu-Film, and Vapor Gard) and surfactants (Tergitol, Zonix, and an unregistered AGAE product) were screened in detached-leaf assays. Anti-Stress, Nu-Film, Zonix, and a Nu-Film-Zonix mixture were additionally tested for durability, protection against exposure to infested water, and a reduction in sporulation. FFP effectiveness was retained for at least 3 weeks of exposure to overhead irrigation and rain. Relative to controls, foliar treatments protected rhododendron branches exposed to infested water. No treatments prevented symptom development when applied postinfection but leaves treated with Anti-Stress, Zonix, and the Nu-Film-Zonix mixture produced significantly fewer sporangia relative to controls. Application of FFPs and surfactants to quarantined, potentially infected plants offers a management tool for reducing infection and sporulation but not symptom expression, thereby limiting disease spread without interfering with disease detection.

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.

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.

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.

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.

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.

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.

Identification and confirmation of downy mildew (Pseudoperonospora cubensis Berk. &amp; Curt.) resistance sources in cucumber (Cucumis sativus L.)

An experiment was designed to identify the resistance source for downy mildew disease in different genotypes of cucumber. Forty-one cucumber genotypes were screened under natural condition and also under artificial epiphytic condition using detached leaf assay method. Genotype IIHR-438 and Cucumis metuliferus L. showed field resistance with an average PDI of 17.66 and 17.46; AUDPC of 772.24 and 764.48, respectively compared to 73.12 PDI and AUDPC of 3096.64 in highly susceptible genotype of IIHR-389. The disease reaction in selected genotypes of cucumber confirmed by artificial screening was in accordance with disease reaction under natural conditions. Resistant genotype IIHR-438 (14.3 PDI) and C. metuliferus L. (12.8 PDI) had least average PDI as compared to susceptible check Swarna Agethi (58.00 PDI) under artificial condition. Statistical analysis of disease severity data over a period for all the forty-one genotypes using non-linear growth model revealed that 99% variability in disease progression. Screening of genotypes under field conditions, sporulation of pathogen, progress of disease, detached leaf assay and non-linear statistical analysis implied that none of genotypes were found to be immune to downy mildew. Wherein the genotype, IIHR-438 and wild cucumber (C. metuliferus L.) were found resistant to downy mildew disease. Hence, it can be utilized in breeding program to develop resistant cultivar in cucumber against Pseudoperonospora cubensis under tropical conditions of India.

Toxin production by melon root rot fungus, Monosporascus cannonballus

A study was conducted on the possible in vitro production of phytotoxins by the melon root rot pathogen, Monosporascus cannonballus. The culture filtrate of a highly virulent isolate of the fungus was tested for its phytotoxic activity on cucumber by detached leaf assay, electrolyte leakage assay and seedling vigour test. In detached leaf bioassay, necrotic symptoms developed on cucumber leaves 5–7 days after application of the culture filtrate. The culture filtrate of M. cannonballus caused substantial reduction in seed germination, vigor of cucumber seedlings and an increase in electrolyte leakage from leaf tissues compared to the control. Studies on the effect of heat and dialysis on the phytotoxic activity of the culture filtrate revealed that the toxic metabolites are thermostable and have low molecular weight (<14 kDa). To purify the toxin, the culture filtrate was extracted with different solvents which were then tested separately for their phytotoxicity. The results revealed that the water fraction after extraction with chloroform retained the highest phytotoxic activity. The toxin was partially purified by thin layer chromatography (TLC) and the fluorescent compound eluted with an Rf value of 0.6 was analyzed by gas chromatography-mass spectrometry (GC-MS) technique. Mass spectral analysis revealed the presence of two metabolites viz., octocrylene and squalene in the toxin fraction.

A new bacterial blight of pomegranate caused by Pseudomonas sp. in Maharashtra, India

In the past few decades, pomegranate production in India has greatly suffered from a highly catastrophic bacterial blight disease caused by Xanthomonas axonopodis pv. punicae. Various management strategies have failed to control the disease. During surveys carried out in Maharashtra, India in 2010–2015, an unusual yellow pigmented bacterium was consistently associated with pomegranate showing blight symptoms. Based on morphological, biochemical, 16S rRNA and gyrase B gene sequencing, the bacterium was identified as Pseudomonas sp. Pathogenicity was assessed both by a detached leaf assay method (in vitro) and in pomegranate plantlets (in vivo). To the best of our knowledge, we report Pseudomonas sp. as a cause of new bacterial blight disease of pomegranate in Maharashtra, India in addition to Xanthomonas axonopodis pv. punicae.

Development of a protocol to phenotype sweet cherry (Prunus avium L.) for resistance to bacterial canker

Suppression of bacterial canker disease of sweet cherry caused by Pseudomonas syringae pv. syringae (Pss), utilizing resistant scion and rootstock varieties holds promise as a cost-effective management strategy. However, a reproducible and rapid method for screening large breeding populations for resistance to Pss poses a challenge. Sweet cherry cultivars Bing, Sweetheart, Regina, Moreau, Emperor Francis and Rainier were used to examine the effects of Pss isolate, inoculum concentration (1 × 102 to 1 × 108 cfu/ml), leaf age (collected from the tip, middle or base of the shoot) and inoculation assay method (attached versus detached leaf) on disease development. Disease severity was influenced significantly (P ≤ 0.05) by inoculum concentration and the virulence of the Pss isolate. An inoculum concentration of 1 × 108 cfu/ml provided the best disease response in both leaf assays and is recommended for disease screening. Also, a significant Pss isolate x cultivar effect was observed suggesting that proper selection of Pss isolate for disease screening is critical. Disease severity was significantly (P ≤ 0.05) greater for newly expanding leaves in detached assays than for leaves classified as young or old. Disease response among cultivars for attached and detached leaf assays was significantly correlated (r = 0.53, P = 0.002), but the detached leaf assay provided better separation in disease severity among cultivars. We conclude that the genotypic variation in disease response among sweet cherry germplasm can be differentiated based on a detached assay using new leaves, a highly virulent Pss isolate, and an inoculum concentration of 1 × 108 cfu/ml.

Prediction of Sporulation and Germination by the Spider Mite Pathogenic Fungus Neozygites floridana (Neozygitomycetes: Neozygitales: Neozygitaceae) Based on Temperature, Humidity and Time.

Neozygites floridana is a pathogenic fungus and natural enemy of the two-spotted spider mite, Tetranychus urticae (Acari: Tetranychidae), which is an important polyphagous plant pest. The aim of this study was to reveal and predict what combination of temperature, relative humidity (RH), and time that enables and promotes primary conidia production and capilliconidia formation in N. floridana (Brazilian isolate ESALQ 1420), in both a detached leaf assay mimicking climatic conditions in the leaf boundary layer and in a semi-field experiment. In the detached leaf assay, a significant number of conidia were produced at 90% RH but the highest total number of primary conidia and proportion of capilliconidia was found at 95 and 100% RH at 25 °C. Positive temperature and RH effects were observed and conidia production was highest in the 8 to 12 h interval. The semi-field experiment showed that for a >90% probability of N. floridana sporulation, a minimum of 6 h with RH >90% and 10 h with temperatures >21 °C, or 6 h with temperatures >21 °C and 15 h with RH >90% was needed. Our study identified suitable conditions for primary- and capilliconidia production in this Brazilian N. floridana isolate. This information provides an important base for building models of a Decision Support System (DSS) where this natural enemy may be used as a tool in Integrated Pest Management (IPM) and a base for developing in vivo production systems of N. floridana.