Detection and Characterization of QoI-Resistant Phytophthora cactorum and P. nicotianae Causing Leather Rot in Florida Strawberry

Phytophthora cactorum and P. nicotianae cause leather rot (LR) of fruit and Phytophthora crown rot (PhCR) in strawberry. LR occurs sporadically but can cause up to 70% fruit loss when weather is conducive. In Florida's annual strawberry winter production system, PhCR can be severe, resulting in plant stunting, mortality, and severe yield loss. Currently, azoxystrobin is labeled for control of LR but not for PhCR. The aims of this research were i) to determine the sensitivity of P. cactorum and P. nicotianae isolates from strawberry to azoxystrobin and ii) to investigate mechanisms of QoI-resistance present in P. cactorum and P. nicotianae based on the known point mutations within the cytb gene. Isolates of both Phytophthora spp. causing LR and PhCR were collected from multiple strawberry fields in Florida between 1997 and 2020. Isolates were tested for sensitivity to azoxystrobin at 0, 0.01, 0.1, 1.0, 10, and 50 µg/ml on potato dextrose agar (PDA) amended with SHAM (100 µg/ml). Isolates were separated into two groups, sensitive isolates, with the 50% effective concentration (EC50) values lower than 1.0 µg/ml, and resistant isolates having EC50 values higher than 50 µg/ml. P. cactorum and P. nicotianae resistance to azoxystrobin was found for isolates collected after 2010. The first 450 nucleotides of the mitochondrial cytochrome b (cytb) gene were sequenced from a selection of resistant and sensitive isolates of both species. The G143A mutation reported to confer resistance to azoxystrobin was found in all resistant P. cactorum isolates. However, in P. nicotianae, qualitative resistance was observed, but the isolates lacked all the known mutations in the cytb gene. This is the first report of resistance to azoxystrobin in P. cactorum and P. nicotianae.

“Shining a LAMP” (Loop-Mediated Isothermal Amplification) on the Molecular Detection of Phytopathogens Phytophthora spp. and Phytophthora cactorum in Strawberry Fields

Phytopathogenic microorganisms belonging to the genus Phytophthora have been recognized many times as causal agents of diseases that lower the yield of many plants important for agriculture. Meanwhile, Phytophthora cactorum causes crown rot and leather rot of berry fruits, mainly strawberries. However, widely-applied culture-based methods used for the detection of pathogens are time-consuming and often inaccurate. What is more, molecular techniques require costly equipment. Here we show a rapid and effective detection method for the aforementioned targets, deploying a simple molecular biology technique, Loop-Mediated Isothermal Amplification (LAMP). We optimized assays to amplify the translation elongation factor 1-α (EF1a) gene for two targets: Phytophthora spp. And Phytophthora cactorum. We optimized the LAMP on pure strains of the pathogens, isolated from organic plantations of strawberry, and successfully validated the assay on biological material from the environment including soil samples, rhizosphere, shoots and roots of strawberry, and with SYBR Green. Our results demonstrate that a simple and reliable molecular detection method, that requires only a thermoblock and simple DNA isolation kit, can be successfully applied to detect pathogens that are difficult to separate from the field. We anticipate our findings to be a starting point for developing easier and faster modifications of the isothermal detection methods and which can be applied directly in the plantation, in particular with the use of freeze-dried reagents and chemistry, allowing observation of the results with the naked eye.

A Comparison of the Ability of Some Commercially Produced Biological Control Agents to Protect Strawberry Plants against the Plant Pathogen Phytophthora cactorum

A comparison of the ability of commercially produced biological control agents—Contans, Gliorex, Hirundo, Polyversum, Prometheus, Clonoplus, Integral Pro and Xilon GR, completed with an isolate of Clonostachys rosea and of Pseudomonas sp.—to protect strawberry plants against Phytophthora cactorum was performed. The experiment was performed on strawberry cultivars Sonata, Karmen, and Wendy—cultivated in a cultivating room and greenhouse. The health of plants was affected negatively by the pathogen in all variants of biological agents used, but differences were seen in the rates of this decrease. The results revealed the ability of some tested agents to improve the growth of plants in the absence of the pathogen; the preparation Polyversum (Pythium oligandrum) was the most beneficial, in both the presence and absence of the pathogen. Contrarily, some agents alone decreased the health of plants; Integral Pro (Bacillus subtillis) and a strain of Pseudomonas sp. caused a deterioration in the health of the plants, even in the absence of a pathogen. The results of our analysis demonstrate the varied usefulness of all agents under unified environmental conditions; their effect seems to be dependent on the conditions and on the combination of the genotypes of all three participants in the interaction: plant–pathogen–antagonist.

Phytophthora cactorum causing bleeding canker of Acer x freemanii in southern Idaho

Since 2018, bleeding cankers have been observed on maple trees in multiple home gardens in southwest Idaho. The cankers ooze a dark sap and and are approximately 10 cm to 35 cm in diameter. Cankers typically occur on the main trunk but are also present on scaffold branches in severe infecrions. Symptoms of foliar chlorois, branch dieback, and premature autumn senescence were also associated with the disease. Phytophthora DNA was detected in symptomatic material from five trees using real-time PCR (Miles et al., 2017). In July 2019 recovery of a causal agent from a symptomatic Acer x freemanii tree was attempted. Excisions were made from the interface of healthy and diseased tissue around the cankers using a chisel. The tissue was then placed in sealed plastic ziplock bags at 4°C for 7 days. Hyphae were then removed with forceps and placed onto potato dextrose agar (PDA) amended with penicillin G (0.2 g/liter) and streptomycin sulfate (0.8 g/liter). Colonies resembling Phytophthora cactorum were consistently observed after 5 days at 21°C. Tentative P. cactorum identification was based on the presence of abundant papillate and caducous sporangia on a short pedicel; sporangia were approximately 30 μm long and 26 μm wide (Bush et al., 2006; Hudler, 2013). Individual hyphal tips were transferred to fresh PDA plates and sequencing of both the rDNA ITS region and Cytochrome c oxidase subunit I (COI) was completed for a representative isolate (D19-130). DNA extraction, PCR and sequencing were as previously described (Woodhall et al. 2013; Robideau et al., 2011). The resulting DNA sequences for rDNA ITS (MW315449) and COI (MW881040) were both 100% identical (723/723 bp and 728/728 bp) with sequences from cultures previously identified as P. cactorum (MH171627 and MH136858). To determine pathogenicity, 14 month-old maple (A. x freemanii) trees in individual containers with potting mix were wounded 15 mm above the soil line with a single 10 mm incision using a sterile razor blade and inoculated by placing a 10 mm2 fully colonized PDA plug of isolate D19-130 on the wound. The inoculum and wound were then covered with a damp cotton ball that was secured loosely with parafilm. Control plants consisted of uninoculated plants and wounded plants inoculated with a PDA agar plug. Each treatment was replicated five times and placed in a controlled environment chamber set at 24ºC and 90% relative humidity. All treatments were sprayed with water daily to ensure the cotton balls remained damp. After 8 weeks, black lesions, up to approximately 25 mm above the soil line, were observed on the stem base of all P. cactorum-inoculated plants. No black lesions were observed on non-inoculated plants or plants inoculated with a PDA agar plug. P. cactorum was isolated from lesions, as described above, except polystyrene foam boxes containing moist paper towels were used instead of bags. This report confirms P. cactorum as a causal agent of bleeding canker of maple in Idaho for the first time. It has been shown that several Phytophthora species can infect maple (Jung and Burgess, 2009; Huddler, 2013). P. cactorum has a wide host range but certain strains have been associated with lethal bleeding stem cankers in maple and other deciduous trees worldwide (Huddler, 2013). Knowledge of the causal agent of bleeding canker on maple will help determine appropriate disease management practices.

Effect of Natural Polysaccharide Matrix-Based Selenium Nanocomposites on Phytophthora cactorum and Rhizospheric Microorganisms

We studied the effects of new chemically synthesized selenium (Se) nanocomposites (NCs) based on natural polysaccharide matrices arabinogalactan (AG), starch (ST), and kappa-carrageenan (CAR) on the viability of phytopathogen Phytophthora cactorum, rhizospheric bacteria, and potato productivity in the field experiment. Using transmission electron microscopy (TEM), it was shown that the nanocomposites contained nanoparticles varying from 20 to 180 nm in size depending on the type of NC. All three investigated NCs had a fungicidal effect even at the lowest tested concentrations of 50 µg/mL for Se/AG NC (3 µg/mL Se), 30 µg/mL for Se/ST NC (0.5 µg/mL Se), and 39 µg/mL for Se/CAR NC (1.4 µg/mL Se), including concentration of 0.000625% Se (6.25 µg/mL) in the final suspension, which was used to study Se NC effects on bacterial growth of the three common rhizospheric bacteria Acinetobacter guillouiae, Rhodococcus erythropolis and Pseudomonas oryzihabitans isolated from the rhizosphere of plants growing in the Irkutsk Region. The AG-based Se NC (Se/AG NC) and CAR-based Se NC (Se/CAR NC) exhibited the greatest inhibition of fungal growth up to 60% (at 300 µg/mL) and 49% (at 234 µg/mL), respectively. The safe use of Se NCs against phytopathogens requires them to be environmentally friendly without negative effects on rhizospheric microorganisms. The same concentration of 0.000625% Se (6.25 µg/mL) in the final suspension of all three Se NCs (which corresponds to 105.57 µg/mL for Se/AG NC, 428.08 µg/mL for Se/ST NC and 170.30 µg/mL for Se/CAR NC) was used to study their effect on bacterial growth (bactericidal, bacteriostatic, and biofilm formation effects) of the three rhizospheric bacteria. Based on our earlier studies this concentration had an antibacterial effect against the phytopathogenic bacterium Clavibacter sepedonicus that causes diseases of potato ring rot, but did not negatively affect the viability of potato plants at this concentration. In this study, using this concentration no bacteriostatic and bactericidal activity of all three Se NCs were found against Rhodococcus erythropolis based on the optical density of a bacterial suspension, agar diffusion, and intensity of biofilm formation, but Se/CAR and AG NCs inhibited the growth of Pseudomonas oryzihabitans. The cell growth was decrease by 15–30% during the entire observation period, but the stimulation of biofilm formation by this bacterium was observed for Se/CAR NC. Se/AG NC also had bacteriostatic and antibiofilm effects on the rhizospheric bacterium Acinetobacter guillouiae. There was a 2.5-fold decrease in bacterial growth and a 30% decrease in biofilm formation, but Se/CAR NC stimulated the growth of A. guillouiae. According to the results of the preliminary field test, an increase in potato productivity by an average of 30% was revealed after the pre-planting treatment of tubers by spraying them with Se/AG and Se/CAR NCs with the same concentration of Se of 0.000625% (6.25 µg/mL) in a final suspension. The obtained and previously published results on the positive effect of natural matrix-based Se NCs on plants open up prospects for further investigation of their effects on rhizosphere bacteria and resistance of cultivated plants to stress factors.

Phosphonate treatment effects on Phytophthora root rot control, phosphite residues and Phytophthora cactorum inoculum in young apple orchards

Phytophthora root rot, caused by Phytophthora cactorum, is an economically important disease on young apple trees. Limited information is available on the effect of different phosphonate application methods and dosages on disease control, fruit- and root phosphite concentrations and soil- and root pathogen inoculum levels. Evaluation of phosphonate treatments in three apple orchard trials (two in the Grabouw and one in the Koue Bokkeveld region) showed that foliar sprays (ammonium- or potassium phosphonate), trunk sprays and trunk paints, were equally effective at increasing trunk diameter in one trial and yield in a second trial over a 25-month period. Foliar ammonium- and potassium phosphonate sprays (12 g phosphorous acid/tree), and two different dosages of the ammonium phosphonate sprays (~ 4.8 g or 12 g phosphorous acid/tree) were all equally effective at improving tree growth. The addition of a bark penetrant (polyether-polymethylsiloxane-copolymer) to trunk sprays did not improve the activity of trunk sprays. The low dosage ammonium phosphonate foliar spray (~4.8 g a.i./tree) was the only treatment that in general yielded significantly lower root phosphite concentrations than the other phosphonate treatments. Root phosphite concentrations were significantly positively correlated (P < 0.0001) with an increase in trunk diameter and negatively with P. cactorum root DNA quantities (P ≤ 0.001). Phosphite fruit residues were less than 31 ppm for all treatments, with the trunk paint treatment (80 g phosphorous acid/tree applied annually) yielding significantly lower residues than the higher dosage foliar sprays (~12 g a.i/tree). Twenty-one months post-treatment, most of the phosphonate treatments in all of the trials similarly significantly reduced P. cactorum DNA quantities estimated directly from roots, but not from soil based on soil baiting DNA analysis. Pathogen quantities in fine feeder roots did not differ significantly from those in higher-order roots (< 5 mm dia.). Phytophthora cactorum DNA quantities estimated using DNA quantification directly from roots were significantly correlated (P < 0.0001) with those obtained through root leaf baiting DNA analysis, and to a lesser extent with soil leaf baiting DNA quantities (P = 0.025).

In Vitro Pathogenesis Caused by Phytophthora cactorum and DNA Analysis of the Strawberry-Resistant Microplants with ISSR Markers

The soil pathogenic fungus Phytophthora cactorum causes the most dangerous diseases occurring in strawberry plantations—strawberry crown rot and leather rot. Modern biotechnology methods, e.g., in vitro culture selection and molecular diagnostics can be utilized in the selection of cultivars that are less susceptible or resistant to Phytophthora diseases. In this study, in vitro selection of four strawberry microclones: ‘Elsanta’, ‘Feltar’, ‘Teresa’ and ‘Plena SVdT’ against Phytophthora cactorum (Lebert and Cohn) J. Schröt was carried out. Molecular analysis with inter simple sequence repeat (ISSR) markers was also used to evaluate genetic similarity of the selected resistant plants. None of the analyzed microclones showed complete resistance to the selection factor, but there were plants in all tested microclones that survived the pressure of the pathogen. Results showed that susceptibility to this pathogenic fungus was significantly differentiated and depended on the microclone. The ‘Feltar’ microclone had the significantly lowest susceptibility to Phytophthora disease, followed by the microclones ‘Elsanta’ and ‘Teresa’ with significantly higher susceptibility. The ‘Plena SVdT’ microclone showed the highest susceptibility to Phytophthora disease. This differentiation was linked to the genetic similarity observed at deoxyribonucleic acid (DNA) level between the resistant plants selected from microclones. Cluster analysis revealed that microclones with similar susceptibility to phytophthorosis, i.e., ‘Elsanta’, ‘Feltar’ and ‘Teresa’, appeared to be genetically similar. The microclone ‘Plena SVdT’ revealed a different course of phytophthorosis from the aforementioned microclones, being the least genetically similar to them.

Rapid and simple detection of Phytophthora cactorum in strawberry using a coupled recombinase polymerase amplification–lateral flow strip assay

Phytophthora cactorum is a devastating pathogen that infects a wide range of plants and causes Phytophthora rot disease, which has resulted in great economic losses in crop production. Therefore, the rapid and practicable detection of P. cactorum is important for disease monitoring and forecasting. In this study, we developed a lateral flow recombinase polymerase amplification (LF-RPA) assay for the sensitive visual detection of P. cactorum. Specific primers for P. cactorum were designed based on the ras-related protein gene Ypt1; all 10 P. cactorum isolates yielded positive detection results, whereas no cross-reaction occurred in related oomycete or fungal species. The detection limit for the LF-RPA assay was 100 fg of genomic DNA under optimized conditions. Combined with a simplified alkaline lysis method for plant DNA extraction, the LF-RPA assay successfully detected P. cactorum in naturally diseased strawberry samples without specialized equipment within 40 min. Thus, the LF-RPA assay developed in this study is a rapid, simple, and accurate method for the detection of P. cactorum, with the potential for further application in resource-limited laboratories.

Phytophthora crown rot of Florida Strawberry: Inoculum Sources and Thermotherapy of Transplants for Disease Management

Phytophthora crown rot, caused mainly by Phytophthora cactorum, and also by the recently reported P. nicotianae, is an important disease in the Florida strawberry annual production system. Mefenoxam is the most effective and widely used fungicide to manage this disease. However, due to pathogen resistance, alternatives to chemical control are needed. Phytophthora spp. were rarely recovered during the summer from soil of commercial farms where the disease was observed during the season. In a more detailed survey on research plots, neither of the two species was recovered one month after the crop was terminated and water was shut off. Therefore, Phytophthora spp. does not seem to survive in the soil over summer in Florida. In a field trial, asymptomatic nursery transplants harboring quiescent infections were confirmed as the major source of inoculum for these pathogens in Florida. Heat treatment of P. cactorum zoospores at 44oC for as little as 5 min was effective in inhibiting germination and colony formation; however, oospore germination was not inhibited by any of the tested temperatures in vitro. In the field, thermotherapy treatment of inoculated plants was shown to have great potential to serve as a non-chemical approach for managing Phytophthora crown rot in production fields and reducing mefenoxam-resistant populations in nursery transplants.