Diversity ofPhytophthora,Pythium,and PhytopythiumSpecies in Recycled Irrigation Water in a Container Nursery

Recycling of irrigation water increases disease risks due to spread of waterborne oomycete plant pathogens such as Phytophthora, Pythium, and Phytopythium. A comprehensive metabarcoding study was conducted to determine spatial and temporal dynamics of oomycete communities present in irrigation water collected from a creek (main water source), a pond, retention reservoirs, a chlorinated water reservoir, and runoff channels within a commercial container nursery in Oregon over the course of 1 year. Two methods, filtration and leaf baiting, were compared for the detection of oomycete communities. Oomycete communities in recycled irrigation water were less diverse but highly enriched with biologically active plant pathogens as compared with the creek water. The filtration method captured a larger portion of oomycete diversity, while leaf baiting was more selective for plant-associated oomycete species of Phytophthora and a few Pythium and Phytopythium species. Seasonality strongly influenced oomycete diversity in irrigation water and detection with leaf baiting. Phytophthora was the major colonizer of leaf baits in winter, while all three genera were equally abundant on leaf baits in summer. The metabarcoding approach was highly effective in studying oomycete ecology, however, it failed to distinguish some closely related species. We developed a custom oomycete internal transcribed spacer (ITS)1 reference database containing shorter sequences flanked by ITS6 and ITS7 primers used in metabarcoding and used it to assemble a list of indistinguishable species complexes and clusters to improve identification. The predominant bait-colonizing species detected in recycled irrigation water were the Phytophthora citricola-complex, Phytophthora syringae, Phytophthora parsiana-cluster, Phytophthora chlamydospora, Phytophthora gonapodyides, Phytophthora irrigata, Phytophthora taxon Oaksoil-cluster, Phytophthora citrophthora-cluster, Phytophthora megasperma-cluster, Pythium chondricola-complex, Pythium dissotocum-cluster, and Phytopythium litorale.

The Use of qPCR Reveals a High Frequency of Phytophthora quercina in Two Spanish Holm Oak Areas

The struggling Spanish holm oak woodland situation associated with Phytophthora root rot has been studied for a long time. Phytophthora cinnamomi is considered the main, but not the only species responsible for the decline scenario. This study verifies the presence and/or detection of Phytophthora species in two holm oak areas of Spain (southwestern “dehesas” and northeastern woodland) using different isolation and detection approaches. Direct isolation and baiting methods in declining and non-declining holm oak trees revealed Phytophthora cambivora, Phytophthora cinnamomi, Phytophthora gonapodyides, Phytophthora megasperma, and Phytophthora pseudocryptogea in the dehesas, while in the northeastern woodland, no Phytophthora spp. were recovered. Statistical analyses indicated that there was not a significant relationship between the Phytophthora spp. isolation frequency and the disease expression of the holm oak stands in the dehesas. Phytophthora quercina and P. cinnamomi TaqMan real-time PCR probes showed that both P. cinnamomi and P. quercina are involved in the holm oak decline in Spain, but P. quercina was detected in a higher frequency than P. cinnamomi in both studied areas. Thus, this study demonstrates that molecular approaches complement direct isolation techniques in natural and seminatural ecosystem surveys to determine the presence and distribution of Phytophthora spp. This is the first report of P. pseudocryptogea in Europe and its role in the holm oak decline should be further studied.

Resistance to Phytophthora Species among Rootstocks for Cultivated Prunus Species

Many species of Phytophthora de Bary are important pathogens of cultivated Prunus L. species worldwide, often invading the trees via their rootstocks. In a series of greenhouse trials, resistance to Phytophthora was tested in new and standard rootstocks for cultivated stone fruits, including almond. Successive sets of the rootstocks, propagated as hardwood cuttings or via micropropagation, were transplanted into either noninfested potting soil or potting soil infested with Phytophthora cactorum (Lebert & Cohn) J. Schöt., Phytophthora citricola Sawada, Phytophthora megasperma Drechs, or Phytophthora niederhauserii Z.G. Abad & J.A. Abad. Soil flooding was included in all trials to facilitate pathogen infection. In some trials, soil flooding treatments were varied to examine their effects on the rootstocks in both the absence and presence of Phytophthora. Two to 3 months after transplanting, resistance to the pathogens was assessed based on the severity of root and crown rot. ‘Hansen 536’ was consistently more susceptible than ‘Lovell’, ‘Nemaguard’, ‘Atlas’, ‘Viking’, ‘Citation’, and ‘Marianna 2624’ to root and/or crown rot caused by P. cactorum, P. citricola, and P. megasperma. By contrast, susceptibility to P. niederhauserii was similarly high among all eight tested genotypes of peach, four genotypes of peach × almond, two genotypes of (almond × peach) × peach, and one genotype of plum × almond. Most plum hybrids were highly and consistently resistant to crown rot caused by P. niederhauserii, but only ‘Marianna 2624’ was highly resistant to both crown and root rot caused by all of the Phytophthora species. The results indicate that there is a broad tendency for susceptibility of peach × almond rootstocks and a broad tendency for resistance of plum hybrid rootstocks to multiple species of Phytophthora.

Draft Genome Sequence of Biocontrol AgentBacillus cereusUW85

Bacillus cereusUW85 was isolated from a root of a field-grown alfalfa plant from Arlington, WI, and identified for its ability to suppress damping off, a disease caused byPhytophthora megaspermaf. sp.medicaginison alfalfa. Here, we report the draft genome sequence ofB. cereusUW85, obtained by a combination of Sanger and Illumina sequencing.

First Report of Phytophthora megasperma Causing Decline and Death on Celtis australis in Italy

European hackberry (Celtis australis L.) is a popular shade tree mainly planted in southern Europe and known to be tolerant to dry and poor soils. In early autumn 2013, hackberry plants grown in soil in a commercial nursery located in the floodplain in Umbria region showed symptoms of wilting, dieback, and death. Up to 100% of the canopy was affected, and over 60% of the plants were symptomatic or dead. A Phytophthora species was consistently isolated from symptomatic 6-year-old plants by plating small pieces of collar and root tissues, cut from the margin of dark-brown necrotic lesions, onto P5ARPH selective medium (4). Pure cultures were obtained by single-hyphal transfers on potato dextrose agar (PDA). Sporangia, produced on pepper seeds in soil extract solution (3), were nonpapillate and noncaducous, measuring 34.0 to 85.0 × 22.0 to 50.0 μm. Oospores had an average diameter of 44 μm with mostly paragynous antheridia. On the basis of morphological features, the isolates were identified as P. megasperma Drech. (2). The identity was confirmed by sequencing the cytochrome c oxidase subunit II (Cox II) (5), which gave 100% identity with P. megasperma sequences available in GenBank (GU222070), and by sequencing the internal transcribed spacer (ITS) using the universal primers ITS4 and ITS6, which gave 99% identity with the AF266794 sequence from Cooke et al. (1). The sequences of one isolate (AB239) were deposited in the European Nucleotide Archive (ENA) with accession numbers HG973451 and HG973450 for Cox II and ITS, respectively. Pathogenicity tests were conducted in the greenhouse with isolate AB239 on eight 2-year-old potted European hackberry plants. Mycelial plugs (5 mm diameter) cut from the margins of actively growing 8-day-old cultures on PDA were inserted through the epidermis to the phloem at the collar level. Two plants were used as controls and treated as described above except that sterile PDA plugs replaced the inoculum. Inoculated plants were kept for 4 weeks in a greenhouse at 24 ± 2°C. During that period, inoculated plants showed wilting symptoms similar to those observed in the field. Lesions were evident at all the inoculation points progressing downward to the roots. Colonies of Phytophthora were isolated from the margins of lesions and identified as P. megasperma, thus fulfilling Koch's postulates. Controls remained symptomless. P. megasperma taxonomy is rather complex since it embraces different subgroups, including host specialized forms (formae speciales), some of which are recognized as biological species. Based on morphological and molecular data presented here, the Phytophthora isolates from hackberry belong to P. megasperma sensu stricto, which is included in the “pathogenic to a broad range of hosts” (BHR) group (1). This pathogen is rather polyphagous, attacking mainly fruit and ornamental woody plants, commonly Prunus spp., Malus spp., and Actinidia deliciosa. Like other homothallic Phytophthora species, it is particularly dangerous due to its abundant production of thick-walled resting oospores that enable long-term survival in the soil. To our knowledge this is the first report of P. megasperma sensu stricto (1) on C. australis and its family Ulmaceae/Cannabaceae. References: (1) D. E. L. Cooke et al. Fungal Genet. Biol. 30:17, 2000. (2) D. C. Erwin and O. K. Ribeiro, American Phytopathological Society, St. Paul, MN, 1996. (3) E. Ilieva et al. Eur. J. Plant Path. 101:623, 1995. (4) S. N. Jeffers and S. B. Martin. Plant Dis. 70:1038, 1986. (5) F. N. Martin and P. W. Tooley. Mycologia 95:269, 2003.