Occurrence of Phytophthora Root and Stem Rot of Kiwifruit in Turkey

Vine decline of kiwifruit was observed in an orchard in Bart?n province of Turkey. Affected vines exhibited poor terminal growth, leaf discoloration and various degrees of dieback, including complete vine death. Symptoms were observed in the field on roots, crowns and stems. Two Phytophthora species were isolated from decayed cortical roots and lower stems of kiwifruits. They were identified as Phytophthora cryptogea and Phytophthora megasperma by their morphological characteristics and the analysis of sequences of the internal transcribed spacer (ITS) region of the rDNA. Pathogenicity of the isolates was tested by stem inoculation on kiwifruit seedlings. After 4 weeks, cankers developed in the plants inoculated with P. cryptogea, while no cankers formed in those inoculated with P. megasperma and in control plants. This is the first report of P. cryptogea causing root and stem rot of kiwifruit in Turkey.     

An in vitro investigation of Malaysian Phytophthora palmivora isolates and pathogenicity study on oil palm

Bud rot disease affecting oil palm in South American countries is reported to be caused by Phytophthora palmivora. P. palmivora is a local pathogen affecting various crops in Malaysia, and this finding caused an alarm, which prompted an investigation of pathogenicity using Malaysian P. palmivora to assess the potentials of this Oomycete to infect oil palm in Malaysia. A total of 11 P. palmivora isolates were obtained from cocoa and durian for the study. Leaf bioassays via artificial inoculation using 50,000 zoospores/ml and mycelial agar disc showed severe necrotic lesions on the infection spot of oil palm (DxP) spear leaves. Mild infection was observed in oil palm spear leaves of OxG hybrids indicating lower susceptibility against P. palmivora infection. Phylogenetic analysis using internal transcribed spacer (ITS) data revealed that Malaysian isolates were genetically similar to Colombian isolates supported by significant bootstrap values. The leaf bioassay results revealed that Malaysian oil palm materials are susceptible towards local P. palmivora infection. The Colombian P. palmivora isolates causing bud rot incidence may have evolved over a long period of time, undergone sequential genetic shift to become more virulent towards Colombian oil palm planting materials.     

Outbreak of Phytophthora cinnamomi causing severe decline of avocado trees in southern Turkey

Since the summer 2017, severe decline symptoms have been observed on 10- to 25-year-old avocado trees in almost all commercial orchards planted in the Mediterranean coastal region of Turkey. Young, newly planted trees in infected orchards were also affected by the disease. Affected trees showed wilting, leaf discoloration, defoliation and severe dieback. Some trees were completely desiccated. Although fine roots of symptomatic trees usually were decayed, reddish brown cankers also occurred on taproots and lateral roots of heavily infected trees. The pathogens were isolated from necrotic root and soil samples of symptomatic trees, using selective medium and soil baiting, and were identified based on morphological features and DNA sequences of the internal transcribed spacer (ITS) region. One isolate each of Phytophthora cryptogea and P. palmivora was identified, while all other isolates were P. cinnamomi. In addition, a subcortical fan-shaped mycelium, characteristic of Armillaria spp., was observed in the stem base of a symptomatic tree and identified as Armillaria gallica by DNA sequences of the internal transcribed spacer (ITS) and the translational elongation factor 1-α (EF 1-α) gene regions. Pathogenicity of Phytophthora isolates was tested by stem inoculation on one-year-old avocado seedlings. Two months after inoculation, canker lesions developed on stems of seedlings inoculated by any of the three Phytophthora spp. In contrast, collenchyma callus formed over the wound points on control plants over the same time period. This is the first report of P. cinnamomi, P. cryptogea, P. palmivora and A. gallica causing root rot of avocado trees in Turkey. In addition, P. cryptogea and A. gallica are reported for the first time associated with disease on this host. Due to the severe symptoms and widespread occurrence, P. cinnamomi should be considered a potential threat to avocado cultivation and natural ecosystems of this region of Turkey.     

Phytophthora cinnamomi Associated with Root and Crown Rot of Walnut in Turkey

Walnut decline caused by Phytophthora sp. occurred in an orchard in Sakarya province in Turkey. Affected young trees showed poor growth, leaf discolouration, root and crown rot and eventual death. A Phytophthora sp. isolated from necrotic taproots and crown tissues. The causal agent of the disease was identified as Phytophthora cinnamomi by morphological characteristics and comparing sequences of internal transcribed spacer (ITS) region. Upon conducting pathogenicity test, averaging 7.8‐cm‐long canker developed on basal stem within 2 weeks, while no cankers developed in the control plants.     

First Report of Phytophthora Foliar Blight on Florida Hopbush (Dodonaea viscosa) in Italy

During 2010, a new foliar blight was detected on potted Dodonaea viscosa cv. Purpurea plants in two nurseries in Catania (Italy). On the basis of morphological and cultural features, the pathogen was identified as Phytophthora palmivora. The internal transcribed spacer (ITS)‐rDNA sequence of a representative Phytophthora isolate from hopbush showed 99% identity with other ITS sequences of different P. palmivora isolates available in GenBank, thus confirming the morpho‐cultural identification. Koch’s postulates were fulfilled by pathogenicity tests on potted D. viscosa cv. Purpurea seedlings. To our knowledge, this is the first report of P. palmivora foliar blight disease on D. viscosa.     

Tuber Blight Development in Potato Cultivars in Response to Different Genotypes of Phytophthora infestans

Migrations or introduction of new genotypes of Phytophthora infestans to a specific region imposes a different perspective for potato production. During 2009–2010, a late blight epidemic affected the Northeastern United States, which quickly spread through several states. The epidemic was characterized by the appearance of a new genotype of P. infestans designated US‐22, which was isolated from tomato and potato. Potato tubers are an essential component of late blight epidemics where the pathogen cannot overwinter on Solanaceous plants. Six potato cultivars were inoculated with 12 isolates of P. infestans (five different genotypes), including isolates of the genotype US‐22. Tuber blight development was characterized in terms of tissue darkening expressed as area under the disease progress curve values and lenticel infection. The responses indicated that US‐8 was more aggressive than US‐22, but US‐22 isolates obtained from potato were more aggressive on potato than those acquired from tomato. Tuber periderm responses to infection were limited, yet US‐8 isolates infected the periderm more often than US‐22 isolates. There were significant differences among the cultivars tested but cv. Jacqueline Lee was the most resistant overall. Although isolates of P. infestans genotype US‐22 were less aggressive in comparison with US‐8 isolates, US‐22 isolates still infected potato tubers and were as aggressive us US‐8 isolates on some cultivars. Management of late blight caused by isolates of US‐22 through host resistance may be feasible but imposes a different set of criteria for consideration from those that US‐8 imposed.     

Rapid detection of Phytophthora nicotianae by simple DNA extraction and real‐time loop‐mediated isothermal amplification assay

Phytophthora nicotianae is a phytopathogenic oomycete with a wide host range and worldwide distribution. Rapid detection and diagnosis at the early stages of disease development are important for the effective control of P. nicotianae. In this study, we designed a simple and rapid loop‐mediated isothermal amplification (LAMP)‐based detection method for P. nicotianae. We tested three DNA extraction methods and selected the Kaneka Easy DNA Extraction Kit version 2, which is rapid and robust for LAMP‐based detection. The designed primers were tested using mycelial DNA from 35 species (81 isolates) of Phytophthora, 12 species (12 isolates) of Pythium, one isolate of Phytopythium and one isolate each from seven other soil‐borne pathogens. All of the 42 P. nicotianae isolates were detected by these primers, and no other isolates gave positive results. Three isolates were tested for the sensitivity of the reaction, and the lowest amounts of template DNA that could be detected were 10 fg for two isolates and 1 fg for the third. The target was detected within 25 min in all tested samples, including DNA extracted from both inoculated and naturally infected plants. In contrast, PCR assays with P. nicotianae‐specific primers failed or showed weakened detection in several samples. Thus, we found that the rapid DNA extraction and LAMP assay methods developed in this study can be used to detect P. nicotianae with high sensitivity, specificity and stability.     

Inducible Proteins in Citrus Rootstocks with Different Tolerance Towards the Root Rot Pathogen Phytophthora palmivora

Activities of defence‐related proteins (β‐1,3‐glucanases, chitinases and peroxidases) and concentrations of total soluble phenolics were measured in roots and leaves of non‐infected and infected plants to investigate the response of different citrus rootstock genotypes to the root rot pathogen Phytophthora palmivora Butler. Infection with the pathogen increased concentrations of total proteins, total phenolics and β‐1,3‐glucanase activity in roots of all genotypes, and increases were associated with the extent of root mass reductions and thus susceptibility of the plants. Root chitinase and root peroxidase levels were slightly reduced or unaltered upon infection. β‐1,3‐Glucanase activity was also elevated in leaves of infected plants, but increases did not differ between tolerant and susceptible rootstocks. Effects of root infection on leaves were typically the reverse of effects on roots for chitinase‐ and peroxidase levels and more pronounced in susceptible rootstock genotypes. Although differences in enzyme expression were observed between susceptible and tolerant citrus seedlings, effects were usually associated with disease progression, and not with resistance to P. palmivora. It is suggested that increased activities of the proteins and soluble phenolics studied are not implicated in the primary defence to Phytophthora root diseases, but may contribute to the inhibition of the pathogen during infection in tolerant citrus.     

Roles of small RNAs in soybean defense against Phytophthora sojae infection

The genus Phytophthora consists of many notorious pathogens of crops and forestry trees. At present, battling Phytophthora diseases is challenging due to a lack of understanding of their pathogenesis. We investigated the role of small RNAs in regulating soybean defense in response to infection by Phytophthora sojae, the second most destructive pathogen of soybean. Small RNAs, including microRNAs (miRNAs) and small interfering RNAs (siRNAs), are universal regulators that repress target gene expression in eukaryotes. We identified known and novel small RNAs that differentially accumulated during P. sojae infection in soybean roots. Among them, miR393 and miR166 were induced by heat‐inactivated P. sojae hyphae, indicating that they may be involved in soybean basal defense. Indeed, knocking down the level of mature miR393 led to enhanced susceptibility of soybean to P. sojae; furthermore, the expression of isoflavonoid biosynthetic genes was drastically reduced in miR393 knockdown roots. These data suggest that miR393 promotes soybean defense against P. sojae. In addition to miRNAs, P. sojae infection also resulted in increased accumulation of phased siRNAs (phasiRNAs) that are predominantly generated from canonical resistance genes encoding nucleotide binding‐leucine rich repeat proteins and genes encoding pentatricopeptide repeat‐containing proteins. This work identifies specific miRNAs and phasiRNAs that regulate defense‐associated genes in soybean during Phytophthora infection.     

Zoospore Production Biology of Pythiaceous Plant Pathogens

Zoospores are major dispersal and infective propagules of pythiaceous species. Built upon a recently developed ‘wet‐plate’ method, the objectives of this study were to develop a better understanding about zoospore production biology. Four broth media and five incubation temperatures were evaluated with 12 isolates of Phytophthora nicotianae and 17 other pythiaceous species in this study. The ‘wet‐plate’ method worked the best for heterothallic species, especially those isolates that do not produce chlamydospores. These species included Phytophthora citrophthora, P. nicotianae, Phytophthora palmivora and Phytophthora tropicalis. They readily produced 10⁵–10⁶ zoospores/ml. Overall, most species and isolates produced more zoospores with 20% clarified V8 broth than the other three media: rye, lima bean and carrot. The optimal temperature for nutrient‐deprived culture without free‐flowing water to produce sporangia typically is 5°C cooler than that for vegetative growth. Fresh and revived cultures are more prolific than those that had been subcultured multiple times. These findings will assist oomycete researchers, adding quality, productivity and efficiency to their future zoospore‐based studies.     

Control of Pratylenchus brachyurus in soybean with Trichoderma spp. and resistance inducers

Trichoderma spp. is a fungus with nematode control potential; however, its potential to control the root lesion nematode Pratylenchus brachyurus remains poorly studied. Thus, the aim of this study was to select Trichoderma spp. isolates and assess their ability to control P. brachyurus in soybean crops. Different experiments were conducted aiming at selecting isolates, assessing whether they were able to reduce nematode penetration in plants or cause mortality in vitro, and whether they were able to induce resistance in soybean, as well as at studying the possibility of using the selected isolates associated with resistance inducers (acibenzolar‐S‐methyl, Ecolife^? and AgroMos^?). The selection experiment found three isolates showing satisfactory results, namely GF422, GF425 and GF427; the GF362 isolate was assessed in the subsequent experiments. These four isolates reduced P. brachyurus penetration in soybean roots and promoted nematode mortality in vitro. Increased total protein and catalase activity were recorded, mainly in the 72‐hr assessments. Overall, the protein production was different between isolates. The best results were found in the combination between the GF362 isolate and the three resistance inducers, between GF427 and Ecolife^?, between GF427 and AgroMos^? and between GF422 and Ecolife^?.     

Morphology,phylogeny, dynamics,and ichthyotoxicity of Pheopolykrikos hartmannii (Dinophyceae) isolates and blooms from New York,USA

We report on morphological observations, phylogenetic analyses, bloom dynamics, and ichthyotoxicity of the common but poorly characterized dinoflagellate Pheopolykrikos hartmannii (Zimmermann) Matsuoka et Fukuyo. From 2008 to 2010 in the Forge River Estuary, NY, USA, P. hartmannii bloomed during summer and early fall, achieving densities exceeding 8,000 cells · mL^?1 and often dominating microphytoplankton communities. Large subunit (LSU) and small subunit (SSU) rDNA sequences demonstrated that NY isolates of P. hartmannii sequences were 99%–100% identical to P. hartmannii isolates from eastern US and Korea. In both the LSU and SSU rDNA phylogenies, the clades containing P. hartmannii sequences were distinct sister clades to those composed of Polykrikos schwartzii and P. kofoidii. In the LSU rDNA phylogeny, however, the clade composed of P. hartmannii and a sequence of the photosynthetic Polykrikos lebourae was well separated from the clade composed of 10 entries of Polykrikos schwartzii and P. kofoidii. In addition, a gap of ~180 bases was observed when the LSU rDNA sequences of P. hartmannii were aligned with P. schwartzii and P. kofoidii but was not observed in the alignment between P. hartmannii and P. lebourae. Using scanning electron microscopy, several morphological features previously not reported for P. hartmannii were observed: a ventral groove located in the sulcus, a deep arc‐like apical concavity within the area of apical groove, scale‐like vesicles, and a shallow, completely enclosed, loop‐like apical groove. Resting cysts with arrow‐like surface spines were produced heterothallically by crossing clonal isolates and germinated single gymnoid cells. Finally, filtered and unfiltered bloom water from the Forge River and clonal cultures of P. hartmannii exhibited acute ichthyotoxicity to juvenile sheepshead minnows (Cyprinodon variegates) and aeration did not mitigate this effect, suggesting P. hartmannii is an ichthyotoxic, harmful alga.     

Biocontrol of Late Blight Disease (Phytophthora capsici) of Pepper and the Plant Growth Promotion by Paenibacillus ehimensis KWN38

For field application of a bacterial strain used to control Phythophthora capsici, we will need a biologically and economically efficient carrier medium. The known antagonist Paenibacillus ehimensisKWN38 was grown in a grass medium where it showed high antifungal and lytic enzyme activities. To demonstrate the potential of P. ehimensisKWN38 for biocontrol of late blight disease in pepper, pot trials were conducted by treating the 1‐month‐old plants with water (W), a selected grass medium (G3), G plus P. ehimensisKWN38 inoculation (G3P) or synthetic fungicide (F). The shoot dry weight in G3P was higher than that in W and F treatments at 15 days after zoospore infection (DZI). The root dry weight in G3P was also higher than that in W. The root mortality of G3 and W increased over 58 and 80% at 15 DZI, and some plants in those treatments wilted due to the failure of root physiology. The plants in G3P and F survived well because of their better root health conditions. Soil cellulase activity of G3P was consistently higher than that of W and F at earlier observation times (0, 2 and 6 DZI). The root β‐1,3‐glucanase activity of G3P promptly increased to maximum shortly after zoospore infection and reached the maximum value of 51.12 unit g^?1 of fresh weight at 2 DZI. All these results indicate that inoculation of P. ehimensisKWN38 to the root zone of potted pepper plants increases plant growth, root and soil enzyme activities and alleviates the root death caused by infection with P. capsici zoospores.     

Mating type and sensitivity of Phytophthora nicotianae from tobacco to metalaxyl and dimethomorph in Henan province,China

Phytophthora nicotianae causes black shank, one of the most important diseases of tobacco worldwide. Metalaxyl and dimethomorph are two fungicides which have been used widely for control of this disease in Henan province, China. A study was conducted to determine the level of metalaxyl and dimethomorph sensitivity in isolates of P. nicotianae from tobacco in Henan province and mating type structure of the pathogen population. A total of 32 isolates were isolated from 11 cities in Henan province. Sensitivity of all isolates to metalaxyl and dimethomorph was tested in vitro, and mating types of all isolates were determined by pairing known A1 and A2 testers. For metalaxyl, EC₅₀ values of 32 P. nicotianae isolates ranged from 0.08 to 2.82 mg/L. Sixteen isolates were sensitive, and the rest were intermediate to metalaxyl. None were classified as resistant isolates. For dimethomorph, EC₅₀ values of 32 P. nicotianae isolates ranged from 0.07 to 0.59 mg/L. All isolates were sensitive to dimethomorph. Thirty‐one isolates were A2 mating type, and one isolate was A0 mating type. No isolate was identified as A1 mating type. These results suggested that the P. nicotianae population in Henan province has already exhibited intermediate resistance to metalaxyl and was still sensitive to dimethomorph, and asexual reproduction was the major form of reproduction for the P. nicotianae population.     

Phenotypic differences among single-oospore cultures of Phytophthora palmivora and P. botryosa from Heavea brasiliensis

Oospores ofPhytophthora palmivora andP. botryosa fromHevea brasiliensis were produced when complementary strains of the same species were incubated on V-8 juice agar in continuous darkness, with or without a subsequent period of continuous light. The oospores germinated at a rate of 15–30 % in demineralised water at 26 °C in normal daylight conditions. Other substrates did not improve the germination rate. Single-zoospore colonies derived from sporangia formed by a single oospore were similar to each other in morphology and in pathogenicity toHevea leaves. Over 400 single-oospore isolates from four intraspecific matings ofP. palmivora, and 102 from one pairing ofP. botryosa, were examined. The progeny differed in morphological appearance, mating behaviour, temperature-growth relations, pathogenicity toHevea leaf petioles and cacao pods, rate of production, shape and size of sporangia and in the abundance of chlamydospores. The progeny from an intraspecific cross ofP. botryosa was more variable, with a few isolates being similar in appearance toP. palmivora, having permanently lost their parental characteristic of producing small oval sporangia in clumps. One isolate in particular was indistinguishable fromP. palmivora in morphology and in its ability to produce functional oospores when mated withP. palmivora. Oospores formed by interspecific crosses could not be germinated. With both species, many progeny was less pathogenic than the parents, and many completely non-infective isolates with respect toHevea, cacao and other host plants were produced. Sexual reproduction resulted in a diversity of phenotypes, and both parental types and recombinants were recovered.     

Parasitic Bacteria and Fungi on Common Mistletoe (Viscum album L.) and Their Potential Application in Biocontrol

This study was carried out to identify pathogenic bacteria and fungi on mistletoe (Viscum album L.) and investigate their potential use in biological control of this parasitic plant. For this purpose, a total of 48 fungal isolate and 193 bacterial strains were isolated from contaminated V. album during the summers 2005–2006. The isolated bacterial strains and fungal isolates were identified by using the Sherlock Microbial Identification System (MIS; Microbial ID, Newark) and microscopic methods, respectively. The bacterial strains that induced hypersensitive reaction (HR) on tobacco (Nicotiana tabacum L.) and fungal isolates were tested for pathogenicity on young shoots of mistletoe by using injection methods. The pathogenic bacterial strains and fungal isolates were also tested for their activity against mistletoe using spray methods. Five bacterial strains (two Burkholderia cepacia, one each of Bacillus megaterium, Bacillus pumilus and Pandoraea pulminicola) were HR and pathogenicity positive when injected but none of them when sprayed on mistletoe. When fungi were injected, 32 isolates were pathogenic but only thirteen when sprayed on mistletoe. Alternaria alternata VA?‐202, VA?‐205, VA?‐217 and Acremonium kiliense VA‐11 fungal isolates were the most effective ones and caused strong disease symptoms on mistletoe. The present study is the first report on the efficiency of potential biocontrol agents against mistletoe in Turkey.     

Root discoloration and shoot symptoms in silver birch after Phytophthora infection in vitro

• There are no records of established plant pathogenic Phytophthora species in Finnish forests, but they are likely in the future. Therefore, the effects of Phytophthora inoculations on young, ca. 2‐month‐old silver birch (Betula pendula) seedling roots and shoots were investigated.
• Visual inspection of dark discoloration, direct PCR and re‐isolation, and detailed root morphology analyses were used to evaluate the effects of Phytophthora inoculation on roots. Symptoms in leaves and stems were also recorded.
• Phytophthora was successfully re‐isolated from 67% of the surface‐sterilized roots of inoculated seedlings, but not from the non‐inoculated control seedlings. Dark discolorations were found more often in the root segments of inoculated seedlings than in control seedlings. In the Phytophthora‐treated seedlings, discoloured root segments were usually linked and found primarily in the main root or lateral roots attached to it, whereas in the control seedlings a few single discoloured root segments were scattered throughout the root systems. The number of root segments was lower in the inoculated than in the control seedlings, indicating root loss after Phytophthora inoculation. In the shoots of inoculated birches, leaf and shoot wilting was observed.
• The appearance of wilting in shoots without visible dark discoloration in the base of stems indicated that symptoms originated from roots inoculated with Phytophthora.

     

Cocoa canker caused by Phytophthora palmivora

Canker disease of Theobroma cacao L. caused by Phytophthora palmivora (Butler) Butler gives rise to serious damage in Fiji and is common in the S.W. Pacific area. Fusarium decemcellulareBiick (imperfect stage of Calonectria rigidiuscula (Berk. & Br.) Sacc.) was constantly associated with P. palmivora in disorders of cocoa but was not found to be a primary pathogen. Most Amelonado cocoa trees were resistant to canker but there were differences in the reactions of other Forastero and of Trinitario types. The F₁ generation of a local Trinitario selection, itself resistant, showed segregation resulting in a proportion of very susceptible plants. Isolates of P. palmivora from black pod, canker and green twigs were equally pathogenic, both to pods and seedling stems. Sporangial dimensions, particularly the length/breadth ratio, differed from those reported elsewhere. It is uncertain whether the reason that cocoa canker is more serious in some countries than others is because of differences in the pathogenicity of P. palmivora strains to bark or the genetic differences of the cocoa grown.