Real-time detection of <Emphasis Type="Italic">Phytophthora nicotianae</Emphasis> and <Emphasis Type="Italic">P. citrophthora</Emphasis>in citrus roots and soil

Two primers, specific for Phytophthora nicotianae (Pn6) and P. citrophthora (Pc2B), were modified to obtain Scorpion primers for real-time identification and detection of both pathogens in citrus nursery soils and roots. Multiplex PCR with dual-labelled fluorogenic probes allowed concurrent identification of both species ofPhytophthora among 150 fungal isolates, including 14 species of Phytophthora. Using P. nicotianaespecific primers a delayed and lower fluorescence increase was also obtained from P. cactorumDNA. However, in separate real-time amplifications, the aspecific increase of fluorescence from P. cactorum was avoided by increasing the annealing temperature. In multiplex PCR, with a series of 10-fold DNA dilutions, the detection limit was 10 pg l^-1 for P. nicotianaeand 100 pg l^–1 for P. citrophthora, whereas in separate reaction DNA up to 1 pg l^-1 was detected for both pathogens.Simple and rapid procedures for direct DNA extraction from soil and roots were utilised to yield DNA whose purity and quality was suitable for PCR assays. By combining these protocols with a double amplification (nested Scorpion-PCR) using primers Ph2-ITS4 amplifying DNA from the main Phytophthora species (first round) and PnB5-Pn6 Scorpion and Pc2B Scorpion-Pc7 (second round), it was possible to achieve real-time detection of P. nicotianaeand P. citrophthora from roots and soil. The degree of sensitivity was similar to that of traditional detection methods based on the use of selective media. The analyses of artificially and naturally infested soil showed a high and significant correlation between the concentration of pathogen propagules and the real-time PCR cycle threshold.     

Collar rot caused byPhytophthora citrophthora on pear trees in Greece

A severe crown rot of pear trees of cultivar ‘Kondoula’ grafted on quince rootstock was observed in Greece. Isolations from the affected tissues repeatadly yielded aPhytophthora sp. that was determined by morphological and physiological characteristics to beP. citrophthora. The pathogenicity of two of theP. citrophthora isolates was tested by inoculating trunks of 2-year-old pear trees by mycelial agar disks. Thirty-two days after inoculation all inoculated trees were infected. Although the pear isolates could not be differentiated from isolates ofP. palmivora orP. nicotianae based on isozyme profiles of α-esterase or lactate dehydrogenase, RAPD profiles with one selected primer differentiated the pear isolates from the other species and revealed an electrophoretic banding pattern similar to that of aP. citrophthora standard. This is the first report ofP. citrophthora on pear trees in Greece.     

Phytophthora boehmeriae boll rot: a new threat to cotton cultivation in the mediterranean region

A boll rot of cotton (Gossypium hirsutum L.) was observed for the first time in Greece in August 1993 in Larissa and Volos counties, and in August and September 1995 in Trikala and Phthiotis counties. Fungi of the genusPhytophthora were isolated from diseased plants. Morphological characteristics of the pathogen were recorded on mounts made directly from the infected tissues or after growth of the isolated fungus on corn meal agar or sterile distilled water. Colony morphology, growth rates, features of asexual and sexual structures and maximum growth temperatures were examined. APhytophthora species new to Europe,Phytophthora boehmeriae Sawada, attacking cotton bolls, was identified. The pathogenicity of the isolates was confirmed by artificial inoculations of detached cotton bolls. Analysis of α-esterase isozymes revealed unique banding patterns for isolates ofP. boehmeriae compared with those ofP. cactorum andP. parasitica, which arePhytophthora species with similar morphology.     

Preliminary evaluation of nine fungicides for control ofPhytophthora cactorum andP. citrophthora associated with crown rot in peach trees

Excised twig assay and excised stem inoculation were used to evaluate nine fungicides (metalaxyl, fosetyl-Al, copper hydroxide, copper sulfate, copper oxychloride, captan, quintozene, propamocarb and chlorothalonil) againstPhytophthora cactorum andP. citrophthora associated with crown rot in peach trees. Segments were soaked in fungicide solutions at different concentrations and then inserted vertically intoP. cactorum orP. citrophthora cultures growing on cornmeal agar plus antibiotics, or inoculated by inserting a mycelium-bearing agar plug directly into the cambium. Following incubation, the bark was scraped off and length of necrosis was measured. Metalaxyl was the only fungicide that inhibited canker development on segments at the manufacturer-recommended concentration. Fosetyl-Al, captan, copper hydroxide and copper sulfate inhibited canker development at 3, 4, 4 and 8 gl^-1, respectively. The other fungicides did not affect canker length significantly compared with non-treated twigs, with the exception of propamocarb, which reduced the development ofP. cactorum on excised stems. The tested methods enabled rapid and effective evaluation of a large number of chemicals to prevent crown rot diseases caused byPhytophthora in the laboratory. http://www.phytoparasitica.org posting Dec. 5, 2001.     

Testing variability in pathogenicity ofPhytophthora cactorum, P. citrophthora andP. syringae to apple, pear, peach, cherry and plum rootstocks

The relative virulence ofPhytophthora cactorum andP. syringae originating from almond trees, and ofP. citrophthora originating from citrus, to apple, pear, peach, cherry and plum rootstocks, was studiedin vivo andin vitro. Results of the different experiments were in good agreement. All testedPhytophthora isolates showed little virulence to pear rootstocks-causing only minor crown rot symptoms - and no virulence at all to apple rootstocks. In contrast, they were highly virulent to stone fruit rootstocks, causing crown rot disease. The non-pathogenicity of these isolates to pome rootstocks could be interpreted as strict host specificity.     

Variation in virulence of Greek isolates ofPhytophthora citrophthora as measured by their ability to cause crown rot on three peach rootstocks

The virulence ofPhytophthora citrophthora isolated from various host-plants on three peach rootstocks (GF677, PR204, KID I) was examined. There was no significant difference among the rootstocks with respect to their susceptibility to testedP. citrophthora isolates. The most virulent isolate originated from sycamore (Acer pseudoplatanus); isolates from pistachio trees (Pistacia vera) also showed high virulence but were significantly less virulent than the sycamore isolate. Isolates originating from plum (Prunus domestica), almond (Prunus amygdalus) and lemon (Citrus limon) trees were moderately virulent on peach rootstocks; those from cyclamen (Cyclamen persicum) showed the lowest virulence of those tested. There was thus great variation in virulence among the testedP. citrophthora isolates. It is possible that the isolates ofP. citrophthora from sycamore, pistachio, plum, almond and lemon trees are a threat to peach trees, whereas the low virulence of the isolates from cyclamen hosts suggests that these pathogens are not a serious threat to peach trees. http://www.phytoparasitica.org posting Jan. 3, 2002.     

Molecular analysis of Phytophthora diversity in nursery‐grown ornamental and fruit plants

The genetic diversity of Phytophthora spp. was investigated in potted ornamental and fruit tree species. A metabarcoding approach was used, based on a semi‐nested PCR with Phytophthora genus‐specific primers targeting the ITS1 region of the rDNA. More than 50 ITS1 sequence types representing at least 15 distinct Phytophthora taxa were detected. Nine had ITS sequences that grouped them in defined taxonomic groups (P. nicotianae, P. citrophthora, P. meadii, P. taxon Pgchlamydo, P. cinnamomi, P. parvispora, P. cambivora, P. niederhauserii and P. lateralis) whereas three phylotypes were associated to two or more taxa (P. citricola taxon E or III; P. pseudosyringae, P. ilicis or P. nemorosa; and P. cryptogea, P. erythroseptica, P. himalayensis or P. sp. ‘kelmania’) that can be challenging to resolve with ITS1 sequences alone. Three additional phylotypes were considered as representatives of novel Phytophthora taxa and defined as P. meadii‐like, P. cinnamomi‐like and P. niederhauserii‐like. Furthermore, the analyses highlighted a very complex assemblage of Phytophthora taxa in ornamental nurseries within a limited geographic area and provided some indications of structure amongst populations of P. nicotianae (the most prevalent taxon) and other taxa. Data revealed new host–pathogen combinations, evidence of new species previously unreported in Italy (P. lateralis) or Europe (P. meadii) and phylotypes representative of species that remain to be taxonomically defined. Furthermore, the results reinforced the primary role of plant nurseries in favouring the introduction, dissemination and evolution of Phytophthora species.     

Intraspecific Variation in Phytophthora citrophthora from Citrus Trees in Eastern Corsica

Isolates of Phytophthora pathogenic to citrus crops on Eastern Corsica and associated with gummosis were identified by PCR-RFLP of internal transcribed spacers (ITS) sequences and characterized by the random amplified microsatellites (RAMS) technique. A sample of 114 isolates collected from diseased trunks and fruits, and from soil, were overwhelmingly Phytophthora citrophthora. Further analysis indicated that the P. citrophthora population was not homogeneous in citrus groves. There were two groups, with a few (4%) atypical isolates in two marginal groups. The major groups have been re-examined in the light of mating behaviour, RFLPs of mitochondrial DNA and sequence comparisons of ITS regions of rDNA. They were found distinct with all these criteria and perhaps constitute distinct taxa. The results indicate that important modifications occurred in the population structure of P. citrophthora over time in Corsican groves. These changes may have impact on the recent outbreaks of gummosis.     

Phytophthora pachypleura sp. nov., a new species causing root rot of Aucuba japonica and other ornamentals in the United Kingdom

Isolates of an unknown Phytophthora species from the ‘Phytophthora citricola complex’ have been found associated with mortality of Aucuba japonica in the UK. Based on morphological characteristics, growth–temperature relationships, sequences of five DNA regions and pathogenicity assays, the proposed novel species is described as Phytophthora pachypleura. Being homothallic with paragynous antheridia and semipapillate sporangia, P. pachypleura resembles other species in the ‘P. citricola complex’ but can be discriminated by its distinctively thick‐walled oospores with an oospore wall index of 0·71. In the phylogenetic analysis based on three nuclear (ITS, β‐tubulin, EF‐1α) and two mitochondrial (cox1, nadh1) DNA regions, P. pachypleura formed a distinct clade within the ‘P. citricola complex’ with P. citricola s. str., P. citricola E and P. acerina as its closest relatives. Phytophthora pachypleura is more aggressive to A. japonica than P. plurivora and P. multivora and has the potential to affect other ornamental species.     

Occurrence and distribution of <Emphasis Type="Italic">Phytophthora</Emphasis> species in European chestnut stands,and their association with Ink Disease and crown decline

The Phytophthora complex associated with Castanea sativa Mill. was investigated in five European countries in 35 regions and with respect to various domestication levels. Annual precipitation and length of drought season were the main parameters that regulated the presence of Phytophthora species in the chestnut stands. Seven species of Phytophthora were detected; three of these, P. megasperma, P. cryptogea and P. syringae had not been previously reported on sweet chestnut. P. cinnamomi. P. cambivora and P. citricola were most frequently isolated. P. cinnamomi and P. cambivora were the species significantly associated with declining trees with symptoms of Ink Disease. P. cinnamomirequired distinct ecological conditions compared to the other species. P. cinnamomi was never detected in sites characterized by minimum temperatures below 1.4 °C, maximum temperature above 28 °C, or soil pH below 5.4. The results obtained provide useful information for modeling the probability of Ink Disease, crown decline and associated Phytophthora species in chestnut groves in global climatic change scenarios.     

Molecular and Pathogenicity Characteristics of Phytophthora nicotianae Responsible for Root Necrosis and Wilting of Pepper (Capsicum annuum L.) in Tunisia

Isolates of Phytophthora from pepper, produced in Tunisia, were characterised according to molecular and pathogenicity criteria. Polymerase chain reaction amplification of the ITS1 region in the ribosomal DNA resulted in different sized fragments. The pepper isolates and P. nicotianae yielded a fragment of 310bp that distinguished it from P. capsici with a fragment of 270bp. The ribosomal RNA gene amplicons of both internal transcribed spacers and the 5.8 S of the pepper Phytophthora and P. nicotianae were digested with 8 endonucleases. The patterns generated, with the 2 enzymes that cut, were identical for both taxa. This molecular analysis corroborated the morphological and biological characteristics and suggests strongly that the isolates of Phytophthora from pepper belong to the species P. nicotianae. Inoculation of pepper, tomato, eggplant and tobacco plants with the isolates of P. nicotianae from pepper showed they were highly pathogenic on pepper but not on tobacco, while their pathogenicity was weak on tomato and eggplant and was associated with atypical symptoms not observed in the field. These pathogenicity tests suggest that pepper isolates of P. nicotianae are particularly adapted to their host and may thus constitute a forma specialis of P. nicotianae.     

A strain ofPseudomonas syringae which does not belong to pathovarphaseolicola produces phaseolotoxin

A bacterial strain, CFBP 3388, isolated from Vetch (Vicia sativa, L.) was identified asP. s. pv.syringae on the basis of nutritional and biochemical patterns which were obtained with classical tests and the Biolog system. It caused necrotic symptoms typical ofP. s. pv.syringae on bean leaves and pods after artificial inoculation. However, the isolate caused a citrulline-reversible inhibition ofE. coli in phaseolotoxin bioassay. Furthermore, with CFBP 3388 DNA as template a 1900 bp DNA fragment, specific for the phaseolotoxin DNA cluster ofP. s. pv.phaseolicola, was amplified by PCR. This is the first demonstration that an isolate ofP. syringae that is not pv.phaseolicola can produce phaseolotoxinAbbreviations bp base pair - kb kilobase - OCT Ornithine Carbamoyl Transferase     

Effects of inoculum density,plant age and temperature on disease severity caused by pythiaceous fungi on several plants

Alfalfa, maize, sorghum and sugarbeet plants were inoculated with zoospores ofPhytophthora andPythium species in order to assess the effects of inoculum density, plant age and temperature on disease severity. Seedlings were grown axenically in test tubes and inoculated with zoospore suspensions. Disease severity was assessed by measuring the root growth and discoloration of treated and control seedlings. The incremental root length of all plants decreased and root discoloration increased as inoculum concentration of the pathogen increased. Changes were more intensive among low levels of zoospore concentrations and no significant differences in disease severity were found for inoculum densities higher than 10⁴ zoospores ml^-1. Disease severity was negatively related to plant age. Disease development on sugarbeet seedlings infected withPythium andPhytophthora species was affected by temperature, but the pattern of response was determined by the pathogen’s temperature preferences. The incremental root length decreased as temperature increased up to 25°C. The effect ofPythium dissimile andPhytophthora cactorum on root length was significantly lower at 35°C than at 25°C, whereasPythium aphanidermatum andPhytophthora nicotianae caused significant damage to roots even at 35°C. http://www.phytoparasitica.org posting Dec. 3, 2001.     

Detection of Phytophthora nicotianae and P. citrophthora in Citrus Roots and Soils by Nested PCR

The polymerase chain reaction (PCR) was used for the specific detection of Phytophthora nicotianae and P. citrophthora in citrus roots and soils. Primers were based on the nucleotide sequences of the internal transcribed space regions (ITS1 and ITS2) of 16 different species of Phytophthora. Two primer pairs, Pn5B–Pn6 and Pc2B–Pc7, were designed specifically to amplify DNA from P. nicotianae and P. citrophthora, respectively. Another primer pair (Ph2–ITS4) was designed to amplify DNA from many Phytophthora species. All primer pairs were assessed for specificity and absence of cross-reactivity, using DNA from 118 isolates of Phytophthora and 82 of other common soil fungi. In conventional PCR, with a 10-fold dilution series of template DNA, the limit of detection was of 1pgl^–1 DNA for all the primer pairs (Ph2–ITS4, Pn5B–Pn6, and Pc2B–Pc7). In nested PCR, with primers Ph2–ITS4 in the first round, the detection limit was of 1fgl^–1 for both the primer sets (Pn5B–Pn6 and Pc2B–Pc7). Simple, inexpensive and rapid procedures for direct extraction of DNA from soil and roots were developed. The method yielded DNA of a purity and quality suitable for PCR within 2–3h. DNA extracted from soil and roots was amplified by nested PCR utilizing primers Ph2–ITS4 in the first round. In the second round the primer pairs Pn5B–Pn6 and Pc2B–Pc7 were utilized to detect P. nicotianae and P. citrophthora, respectively. Comparison between the molecular method and pathogen isolation by means of a selective medium did not show any significant differences in sensitivity.     

Effect of different hosts onThaumetopoea pityocampa populations in northeast Portugal

A study was carried out at the Natural Park of Montesinho, NE Portugal, in order to evaluate the effect of different pine species (Pinus pinaster Ait.,P. nigra Arn. andP. sylvestris L.) onThaumetopoea pityocampa populations. The structure of the egg batches, the impact of the egg parasitoids on natural mortality of the pest and the species of parasitoids present, as well as their emergence dynamics, were analyzed. The length of the egg batches varied among pine species with the longest ones onP. nigra. The mean number of eggs per batch differed betweenP. sylvestris and the two other hosts studied, with fewer eggs per batch on the first. No differences were found in the size of eggs among pine species. The egg mortality varied between 25.8% and 33.0%, with no differences among hosts. Parasitism was the main cause of death.Baryscapus servadeii (Mercet.) was the most abundant parasitoid species, followed byOoencyrtus pityocampae (Dom.) andTrichogramma embryophagum Htg.B. servadeii dominated in the egg batches collected fromP. pinaster andP. nigra, whereasO. pityocampae was most frequent onP. sylvestris. The emergence ofB. servadeii started in the middle of March and continued until August, with the emergence peak at the end of May. The emergence ofO. pityocampae started at the end of April and continued throughout September, with maximum values in June. http://www.phytoparasitica.org posting Sept. 20, 2006.     

Stone Fruit Tree Apoplexy Caused by Phytophthora Collar Rot

Stone fruit tree apoplexy in Greece is often caused by Phytophthora spp. Two types of Phytophthora apoplexy of stone fruit tree can be distinguished, one occurring during the hot summer period and usually caused by P. cactorum (Leb. et Cohn) Schröt. or P. citrophthora (R. et E. Smith) Leonian, and a second occurring in winter or early spring, and caused by P. syringae (Kleb.) Kleb. or sometimes P. megasperma Drechsler. Inoculation tests showed P. syringae to be highly pathogenic to apricot and almond, whereas certain clonal rootstock material showed resistance. Measures of control against Phytophthora attacks are given.     

Phytophthora root rot of sweet pepper

Phytophthora capsici proved to be the causal agent of a root and crown rot of sweet pepper in the Netherlands.P. capsici was pathogenic on sweet pepper, tomato and sometimes on eggplant but not on tobacco Xanthi. Of these test plants only tomato was infected byP. nicotianae.No different symptoms in plants infected with eitherP. capsici orP. nicotianae were found. Dipping the roots of tomato and sweet pepper plants in a suspension ofP. capsici resulted in a more severe attack than pouring the suspension on the stem base.Resistance in tomato toP. nicotianae did not include resistance toP. capsici. A method to distinguishP. capsici fromP. nicotianae after isolation from soil is described. Both species were able to infect green fruits of tomato and sweet pepper.p. capsici survived in moist soil in the absence of a host for at least 15 months.Samenvatting Phytophthora capsici bleek de oorzaak te zijn van een voet-en wortelrot in paprika op twee bedrijven in 1977 in Nederland.P. capsici was pathogeen op paprika, tomaat en soms op aubergine maar niet op tabak Xanthi.P. nicotianae tastte van deze toetsplanten alleen tomaat aan. Verschillen in symptomen tussenP. nicotianae enP. capsici werden bij tomaat niet waargenomen.Het dompelen van de wortels in eenP. capsici suspensie gaf een ernstiger aantasting dan het begieten van de wortelhals met deze suspensie.Resistentie in tomaat tegenP. nicotianae bleek geen resistentie tegenP. capsici in te houden. P. capsici kan in grond worden aangetoond door groene paprikavruchten als vangsubstraat te gebruiken.P. capsici enP. nicotianae kunnen beide zowel vruchten van tomaat als paprika aantasten. P. capsici overleefde een periode van 15 maan den in vochtige grond waarop geen waardplant werd geteeld.     

New Ichneumonidae (Hymenoptera) parasitoids of forest insect pests in Bulgaria

During the period 1989–1998 investigations were carried out on the parasitoids of some forest insect pests in Bulgaria. Twenty-one ichneumonid species are reported for the first time in Bulgaria as parasitoids of different coleopteran, lepidopteran, and hymenopteran hosts. From these, 6 parasitoids are new records for the parasitoid complexes of the hosts:Exochus decoratus Holmgr. onEudemis profundana (Den. & Schiff.) andGelechia turpella (Den. & Schiff.);Lissonota culiciformis Grav.—onParanthrene tabaniformis (Rott.);Lissonota unicincta Holmgr.—onG. turpella; Xorides gracilicornis (Grav.)—onXylotrechus sp.; andEriborus terebrator Aubert—onClostera anastomosis (L.). Most parasitoids developed as primary parasitoids in the hosts.Itoplectis alternans (Grav.) is a primary parasitoid ofNycteola asiatica (Krul.) and a hyperparasitoid ofE. terebrator: Acropimpla pictipes (Grav.) is secondary parasitoid onAnacampsis populella (Cl.). With the exception of two species ofOlesicampes genus, which destroyed over 50% ofStauronematus compressicornis (F.) andPristophora conjugata (Dahlm.) larvae, andE. terebrator which killed 15.4% ofC. anastomosis population, the rest of the parasitoids occurred in low densities, and did not play an important role in reducing the number of their hosts.     

<Emphasis Type="Italic">Pythium</Emphasis> and <Emphasis Type="Italic">Phytophthora</Emphasis> species associated with root and stem rots of kalanchoe

Pythium and Phytophthora species were isolated from kalanchoe plants with root and stem rots. Phytophthora isolates were identified as Phytophthora nicotianae on the basis of morphological characteristics and restriction fragment length polymorphism (RFLP) analysis of the rDNA-internal transcribed spacer regions. Similarly, the Pythium isolates were identified as Pythium myriotylum and Pythium helicoides. In pathogenicity tests, isolates of the three species caused root and stem rots. Disease severity caused by the Pythium spp. and Ph. nicotianae was the greatest at 35°–40°C and 30°–40°C, respectively. Ph. nicotianae induced stem rot at two different relative humidities (60% and >95%) at 30°C. P. myriotylum and P. helicoides caused root and stem rots at high humidity (>95%), but only root rot at low humidity (60%).     

Prevalence of Phytophthora species in macadamia orchards in Australia and their ability to cause stem canker

In Australia, Phytophthora cinnamomi is the only species reported as the causal agent of stem canker and root rot in macadamia. In other countries, five Phytophthora species have been reported to cause diseases in macadamia, which led us to question if more than one Phytophthora species is responsible for poor tree health in macadamia orchards in Australia. To investigate this, samples were collected from the rhizosphere, stem, and root tissues of trees with and without symptoms, nurseries, and water sources from 70 commercial macadamia orchards in Australia. Phytophthora isolates were identified based on morphological characteristics and DNA sequencing. P. cinnamomi was the most predominant and widely distributed species, and was obtained from the different types of samples including symptomless root tissues. In addition to P. cinnamomi, only P. multivora was isolated from diseased tissue (stem canker) samples. Six other Phytophthora species were obtained from the rhizosphere samples: P. pseudocryptogea, P. citrophthora, P. nicotianae, P. gondwanense, P. sojae, and a new Phytophthora taxon. Only P. cinnamomi was obtained from macadamia nursery samples, while five Phytophthora species were obtained from water sources. Of the heterothallic Phytophthora species, mating type A2 isolates were dominant in P. cinnamomi isolates, whereas only mating type A1 isolates were obtained for P. nicotianae, P. pseudocryptogea, and P. citrophthora. Pathogenicity assays revealed that P. cinnamomi and P. multivora caused significantly larger stem and leaf lesions than P. citrophthora, P. nicotianae, and P. pseudocryptogea. Phytophthora sp. and P. sojae were nonpathogenic towards leaves and stems.