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.     

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.     

Control of phytophthora gummosis of citrus with systemic fungicides in Brazil1

Phytophthora gummosis and phytophthora root rot are the most important fungal diseases of citrus in the State of São Paulo, Brazil. Phytophthora citrophthora and P. nicotianae var. parasitica are the main species that incite both diseases in all the citrus-growing areas of the State. Studies on the control of gummosis and root rot with systemic fungicides have been under way in the State since 1980. The efficacy of fosetyl-A1 and metataxyl to control gummosis incited by both fungi in lemon and sweet orange trees was confirmed. The best control was achieved when foliar sprays of fosetyl-A1 were combined with soil applications of metataxyl. The best timing was determined for application of these systemic products to control gummosis in the state.     

Present status of Phytophthora species in the Mediterranean area,especially in relation to citrus1

Of the 44 currently accepted species of Phytophthora, 18 have been reported in the Mediterranean area. The status of each is briefly reviewed. On citrus, P. citrophthora and P. nicotianae var. parasitica are mainly responsible for foot rot and gummosis, and in addition P. hibernalis, P. syringae, P. cifricola and P. cactorum for brown rot of fruits. The incidence of these species is closely linked to their temperature requirements. The use of sour orange as a resistant rootstock has long provided satisfactory control of foot rot and gummosis, while chemical treatments are effective against brown rot. However, there are indications that, with changingcultural practices, the resistance of sour orange is less well expressed, and the species is incidentally also susceptible to citrus tristeza closterovirus. Alternative rootstocks are therefore being sought.     

DNA sequence analysis of ribosomal ITS region 1 of Phytophthora vignae f. sp. adzukicola, the pathogen that causes stem rot of adzuki bean

Sequences of the internal transcribed spacer (ITS) region 1 were used to examine the phylogenetic relationships among races of 19 isolates of Phytophthora vignae f. sp. adzukicola and between this forma specialis and three isolates of the closely related P. vignae f. sp. vignae. The ITS 1 sequences were highly conserved (> 98.7% similarity) among representatives of both formae speciales groups. The results of this study indicate that P. vignae is a monophyletic group. The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession nos. AB120062–AB120080 and AB120122     

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.     

Phytophthora root and crown rot of raspberry in Bulgaria

Root and crown rot of raspberry (Rubus idaeus L.) was observed in a plantation at the experimental station of small fruits in Kostinbrod, Bulgaria. Isolates ofPhytophthora spp. were obtained from diseased plants. Colony morphology, growth rates, features of asexual and sexual structures were studied and as a result twoPhytophthora species were identified:Phytophthora citricola Saw. andPhytophora citrophthora (R.E. Sm. & E.H. Sm.) Leonian. Their pathogenicity was confirmed in artificial inoculation experiments. The isozyme (-esterase) patterns ofP. citrophthora andP. citricola isolates from raspberry and from the collection of the CBS, Baarn the Netherlands were compared, using micro-gel electrophoresis. Both species are reported for the first time as pathogens of raspberry in Bulgaria. This is only the second report in phytopathological literature ofP. citrophthora on raspberry, the first being from Chile [Latorre and Munoz, 1993].     

Genetic variation and host specificity of <Emphasis Type="Italic">Phytophthora citrophthora</Emphasis> isolates causing branch cankers in Clementine trees in Spain

Considerable tree losses have been observed during the past few years in Spain due to Phytophthora branch canker of clementines caused by Phytophthora citrophthora. The emergence of this disease led to the speculation that either the pathogen has evolved increasing its aggressiveness or specificity to clementines. A total of 134 isolates of P. citrophthora collected from 2003 to 2005 in 135 citrus orchards in Spain and 22 reference isolates were analyzed genotypically and phenotypically to determine the structure of the population. Genotypic diversity was evaluated by means of Inter-Simple Sequence Repeat (ISSR) markers. Among the phenotypic characteristics examined, sporangial characters, sexual behavior, growth rates and colony morphology of the isolates at different temperatures were studied. The aggressiveness and host-specificity of selected isolates were evaluated by pathogenicity tests on sweet oranges and clementines under field conditions. Phytophthora branch canker of clementines was associated mainly with one genotype (P-1), which included 88% of the isolates obtained from branches. Strains isolated years before the first disease outbreak clustered also with this major genotype, thus it may be considered as a predominant population. Thirteen other minor genotypes were determined, but most contained only one isolate. Although there was wide variation in the morphological and physiological characters, all Phytophthora isolates obtained from branch cankers were sexually sterile and showed a characteristic petalloid colony pattern. As in previous greenhouse studies, pathogenicity tests under field conditions demonstrated that clementines and their hybrids were more susceptible to P. citrophthora than sweet oranges. However, no evidence was found to support the hypothesis that the emergence of the disease was associated with more aggressive or host-specific forms of P. citrophthora.     

Ribosomal DNA Sequence Comparisons of Colletotrichum Graminicola from Turfgrasses and other Hosts

The 5.8S ribosomal gene and the flanking internal transcribed spacers (ITS) 1 and 2 from Colletotrichum graminicola isolates causing anthracnose disease of Agrostis palustris and Poa species were sequenced. Although bootstrap support was not high, two major groups were observed with both UPGMA and parsimony algorithms, one containing isolates from A. palustris and another with isolates from Poa spp. The ITS sequences were also compared with those of isolates of C. graminicola and C. sublineolum from Sorghum spp., Zea mays and Rottboellia cochinchinesis as well as other Colletotrichum species. Except for one isolate from P. annua in Texas, the ITS1 and ITS2 sequences of turfgrass isolates always grouped separately from C. graminicola or C. sublineolum from non-turfgrass hosts with high bootstrap support. ITS sequences of the turfgrass isolates were more similar to those of other species of Colletotrichum, such as C. coccodes and C. dematium, than they were to C. graminicola isolates from other hosts. Turfgrass isolates have ITS sequences which are not identical to those of isolates from Zea mays and Sorghum species demonstrating diversity among fungi conventionally classified as C. graminicola.     

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.     

Two subpopulations of Colletotrichum acutatum are responsible for anthracnose in strawberry and leatherleaf fern in Costa Rica

Strawberry, Fragaria × ananassa, and leatherleaf fern, Rumohra adiantiformis, are two important crops in Costa Rica. One of the most severe diseases affecting these crops is anthracnose, caused by members of the fungal genus, Colletotrichum (teleomorph; Glomerella). Eighty single-spore isolates from strawberry and leatherleaf fern were identified as Colletotrichum acutatum by species-specific PCR, and were further characterised by Universally Primed PCR (UP-PCR) fingerprinting analysis, and sequence analysis of the ribosomal internal transcribed spacer (ITS) region. Morphological differences, genotypic variation revealed by UP-PCR fingerprinting analysis, and a single sequence polymorphism within the ITS2 region were found between the isolates from strawberry and leatherleaf fern, respectively. The UPGMA cluster analysis of the fingerprints clearly separated the isolates derived from strawberry and leatherleaf fern into two different clusters. Pathogenicity assays on detached strawberry fruits confirmed the apparent difference between the two groups of isolates. It is therefore suggested that the pathogens responsible for strawberry anthracnose fruit rot and leatherleaf fern anthracnose in Costa Rica, belong to two distinct subpopulations of C. acutatum.     

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.     

Discrimination of <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> isolates from sweet and wild cherry using rep-PCR

Bacterial canker is one of the most important diseases of cherry (Prunus avium). This disease can be caused by two pathovars of Pseudomonas syringae: pv. morsprunorum and pv. syringae. Repetitive DNA polymerase chain reaction-based fingerprinting (rep-PCR) was investigated as a method to distinguish pathovars, races and isolates of P. syringae from sweet and wild cherry. After amplification of total genomic DNA from 87 isolates using the REP (repetitive extragenic palindromic), ERIC (enterobacterial repetitive intergenic consensus) and BOX primers, followed by agarose gel electrophoresis, groups of isolates showed specific patterns of PCR products. Pseudomonas syringae pv. syringae isolates were highly variable. The differences amongst the fingerprints of P. syringae pv. morsprunorum race 1 isolates were small. The patterns of P. syringae pv. morsprunorum race 2 isolates were also very uniform, with one exception, and distinct from the race 1 isolates. rep-PCR is a rapid and simple method to identify isolates of the two races of P. syringae pv. morsprunorum; this method can also assist in the identification of P. syringae pv. syringae isolates, although it cannot replace inoculation on susceptible hosts such as cherry and lilac.     

Ribotyping of EntomopathogenicVerticittium lecanii in Japan

To estimate the genetic diversity in 30 isolates ofVerticillium lecanii from aphids, whiteflies, mite and black pine in Japan, including two commercialized strains (Mycotal and Vertalec), DNA polymorphisms in ribosomal DNA of those isolates were analyzed using polymerase chain reaction (PCR). The internal transcribed spacer (ITS) and intergenic spacer (IGS) regions of the nuclear ribosomal RNA gene of each isolate were analyzed by PCR-RFLP (restriction fragment length polymorphism). The size of the PCR product from the ITS region was ~ 580 bp in 27 of the isolates. A 600 bp ITS product was detected in Mycotal and Vertalec. One Japanese isolate produced both the 580 bp and 600 bp products. Enzymatic digestion of the ITS region with Sau3A I,Msp I,Hae III andRsa I revealed RFLPs that consisted of eight haplotypes. Mycotal and Vertalec were specific haplotypes that differed from other isolates. The Japanese isolates had a complex relationship with the original host, but we identified several specific haplotypes common to an aphid origin. Ten distinct IGS haplotypes were detected in the IGS region, some of which were associated with aphid and whitefly origins. These results suggest that the haplotype of rDNA RFLP analysis can be used for studying genetic diversity inV. lecanii.     

Molecular and Pathotype Analysis of the Rice Blast Fungus in North Vietnam

Rice blast caused by Pyricularia oryzae is a devastating disease worldwide. In Vietnam, rice blast is especially severe in the Red River Delta in the North. The genetic diversity of 114 P. oryzae isolates collected from rice in 2001 in the Red River Delta and nine additional Vietnamese P. oryzae isolates was analysed using Amplified Fragment Length Polymorphism (AFLP). DNA similarity and cluster analysis based on 160 polymorphic AFLP markers showed twelve different AFLP genetic groups among the 123 field isolates. Isolates collected from japonica hosts clustered separately from indica host isolates with at least 60% dissimilarity with little evidence for gene flow between the two populations. In the 2001 population originating from indica hosts, three genetic groups were predominant and represented 99% of the isolates sampled. One predominant clonal lineage represented 59% of the 2001 indica host population and was found in eleven provinces in the Red River Delta of North Vietnam. Significant genotype flow could be demonstrated between the indica population south of Red river and the indica population north of Red river. There was significant linkage disequilibrium between the AFLP loci within the indica population, indicating that this is not a random mating population. Pathogenicity tests of 25 isolates selected from the 12 AFLP groups on a set of 29 differential rice lines revealed two avirulent isolates and 23 pathotypes. Different combinations of known resistance genes were found to have potential for blast resistance breeding for North Vietnam. First two authors contributed equally     

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.     

Population structure of <Emphasis Type="Italic">Eleusine</Emphasis> isolates of <Emphasis Type="Italic">Pyricularia oryzae</Emphasis> and its evolutionary implications

Population structure of Eleusine isolates of Pyricularia oryzae (Magnaporthe oryzae) was examined using DNA markers. On the basis of rDNA sequences, Eleusine isolates were divided into two groups. One group clustered with Triticum isolates, while the other clustered with Eragrostis isolates. This grouping was supported by DNA fingerprinting with three repetitive elements: MGR586, MGR583, and grasshopper. These results suggest that the population of Eleusine isolates is composed of at least two groups that evolved independently from the original population of P. oryzae. Most of the isolates that were collected just after an outbreak of finger millet blast in the 1970s had almost identical fingerprint profiles although they were collected in distant prefectures. This result supports the idea that the outbreak was caused by seed transmission of a particular strain of Eleusine isolates.     

Seasonal susceptibility of citrus scions to <Emphasis Type="Italic">Phytophthora citrophthora</Emphasis> and <Emphasis Type="Italic">P. nicotianae</Emphasis> and the influence of environmental and host-linked factors on infection development

Three citrus scions were evaluated to determine seasonal changes in susceptibility to infections by Phytophthora citrophthora and Phytophthora nicotianae. In a period of 24 months, the Clementine mandarin cv. Hernandina, the hybrid Fortune mandarin and the sweet orange cv. Lane-Late were branch-inoculated under field and laboratory conditions. Field studies showed that the cultivars inoculated with P. citrophthora developed the highest lesion areas during March–June (spring) and September–October (autumn) and with P. nicotianae from June to August (summer). However, lesion areas on detached citrus branches did not show a definite pattern of infection because lesion sizes fluctuated irregularly during the study. The lesion area caused by P. nicotianae in different citrus scions correlated significantly with the monthly mean maximum values of temperature, relative humidity, and the percentage of the relative water content in the 24-month period of inoculations. In contrast, there was no correlation between these variables and the extent of colonisation by P. citrophthora. Nevertheless, a significant relationship was observed between lesion areas caused by P. citrophthora from October to May of each year and the same variables that were significant in inoculations with P. nicotianae. Seasonal changes in the susceptibility of citrus cultivars to P. citrophthora and P. nicotianae may facilitate timing of disease control measures to coincide with periods when disease development is greatest.     

A new <Emphasis Type="Italic">Rhizoctonia</Emphasis> sp. closely related to <Emphasis Type="Italic">Waitea circinata</Emphasis> causes a new disease of creeping bentgrass

Isolates of an unidentified Rhizoctonia sp. (UR isolates) were obtained from creeping bentgrass and Kentucky bluegrass with reddish brown sheath and foliar rots. Because the UR isolates anastomosed with isolates of three varieties of Waitea circinata (var. oryzae, var. zeae, and var. circinata), colony morphology, hyphal growth rate at different temperatures, pathogenicity, sequence analysis of the internal transcribed spacers (ITS) region of ribosomal RNA genes (rDNA) were compared. The colony color of mature UR isolates was distinct from isolates of the other three varieties of W. circinata. In pathogenicity tests on creeping bentgrass, the severity of the disease caused by UR isolates was significantly higher than that caused by the three varieties of W. circinata. Sequence similarities of the rDNA-ITS region between UR isolates and between isolates within each variety were high (97–100%), but they were lower among isolates from UR and the varieties of W. circinata (88–94%). In a phylogenetic tree based on the rDNA-ITS sequences, UR isolates formed a cluster separate from each of the clusters formed by the three varieties of W. circinata. These results indicate that the UR isolates clearly differ from the three varieties of W. circinata. We therefore propose that the UR isolates be classified as new Rhizoctonia sp. that are closely related to W. circinata and that the disease on creeping bentgrass should be called Waitea reddish-brown patch disease (Sekikasshoku-hagusare-byo in Japanese).     

Identification of two serological flagellar types (H1 and H2) inPseudomonas syringae pathovars

Flagellar antigen specificity was studied for the speciesPseudomonas syringae, P. viridiflava andP. cichorii. After checking their motility, bacteria were reacted against six polyclonal antisera containing anti-O (LPS) and anti-H (flagellar) antibodies by indirect immunofluorescent staining. Two distinct flagellar serotypes (H1 and H2) were described. The distribution of H1 and H2 serotypes was then determined for a collection of 88 phytopathogenicPseudomonas strains. Serotype H1 was possessed byP. syringae pv.aptata (12 strains),P. s. pv.helianthi (2),P. s. pv.pisi (11), andP. s. pv.syringae (13). Serotype H2 was possessed byP. cichorii (2),P. s. pv.delphinii (1),P. s. pv.glycinea (4),P. s. pv.lacrymans (1),P. s. pv.mori (1),P. s. pv.morsprunorum (10),P. s. pv.persicae (1),P. s. pv.phaseolicola (8),P. s. pv.tabaci (10) andP. s. pv.tomato (1).P. viridiflava (5) revealed HI, H2 and untyped flagella. The following isolates were untypable by the H1/H2 system:P. corrugata (3),P. fluorescens (2),P. tolaasii (1). H1/H2 serotypes distribution is not linked toP. syringae O-serogroups. On the other hand, H1/H2 distribution seems remarkably linked to the new genospecies of theP. syringae group.Abbreviations CFBP French Collection of Phytopathogenic Bacteria, Angers, France - ICMP International Collection of Micro-organisms from Plants, Auckland, New-Zealand - NCPPB National Collection of Plant Pathogenic Bacteria, Harpenden, Great Britain