Phytophthora mississippiae sp. nov., a New Species Recovered from Irrigation Reservoirs at a Plant Nursery in Mississippi

The genus of Phytophthora was first described by Heinrich Anton de Bary in 1876 [1]. “Phytophthora” was from the Greek word “φυτόνφθορά” which means “the plant-destroyer”. The name evidences that Phytophthora genus includes a group of destructive plant pathogens. This genus was divided into 10 clades following phylogenetic analyses [2-4]. Among these clades, clade 6 has a strong association with forest and riparian environments [5]. It currently consists of 18 formal species. All were described after the year 2000, except for P. gonapodyides [6], P. humicola [7] and P. megasperma [8]. Clade 6 also includes a number of provisional species such as P. taxon forest soil, P. taxon oaksoil, and P. taxon Pgchlamydo [9], as well as many other undescribed taxa (Hong et al. unpublished).


Introduction
The genus of Phytophthora was first described by Heinrich Anton de Bary in 1876 [1]. "Phytophthora" was from the Greek word "φυτόνφθορά" which means "the plant-destroyer". The name evidences that Phytophthora genus includes a group of destructive plant pathogens. This genus was divided into 10 clades following phylogenetic analyses [2][3][4]. Among these clades, clade 6 has a strong association with forest and riparian environments [5]. It currently consists of 18 formal species. All were described after the year 2000, except for P. gonapodyides [6], P. humicola [7] and P. megasperma [8]. Clade 6 also includes a number of provisional species such as P. taxon forest soil, P. taxon oaksoil, and P. taxon Pgchlamydo [9], as well as many other undescribed taxa (Hong et al. unpublished).
Several factors have contributed to the recent increase in the number of species in clade 6. First, advancements in molecular biology and sequence analysis provide viable alternatives to morphospecies concepts used in traditional taxonomic systems such as the taxonomy key of Phytophthora species developed by Waterhouse [10]. Accompanying these advancements is identification of definitive characters and phylogenetic analysis tools that have greatly facilitated re-examination of Phytophthora collections and description of new species. For example, P. rosacearum and P. sansomeana were recently separated from the P. megasperma species complex after sequence analyses [11]. P. sp. O-group isolated in the 1990s was formally named as P. inundata [12]. P. taxon Salixsoil first isolated in the 1970s was assigned as P. lacustris [13]. Many newly isolated species in clade 6 such as P. bilorbang [14] and P. gemini [15] were also described by taking advantage of phylogenetic analysis. Second, recent occurrence of sudden oak death (SOD) caused by P. ramorum in the United States [16,17] and forest declines caused by several Phytophthora species in other countries [18,19] has motivated global surveys of natural habitats and waterways for these pathogens. These surveys done in natural environments recovered a number of new species such as P. borealis and P. riparia [20], plus other taxa that belong to clade 6. Third, parallel surveys of irrigation systems have been greatly intensified to address growing concerns over the increasing Phytophthora disease risk as agricultural industries increasingly use recycled water in the light of global water scarcity [21,22]. The surveys in irrigation systems also recovered a number of novel Phytophthora species [23][24][25] and many new taxa in clade 6 (Hong et al. unpublished).
The objective of this study was to characterize and describe a group of isolates belonging to a previously unknown Phytophthora species recovered from irrigation reservoirs in Mississippi. We describe the morphological, physiological and molecular characters of this new taxon and formally name it as Phytophthora mississippiae sp. nov.

Isolation and isolate maintenance
The origin of four Phytophthora mississippiae isolates examined in this study is shown in Table 1. They were recovered from the surface, middle, or bottom of water columns in irrigation reservoirs at an ornamental plant nursery of Mississippi in 2012 by baiting with rhododendron leaves [26,27]. These baits were deployed in the surveyed reservoirs for 7 days then transferred to a laboratory. They were then cut into approximately 3×3 cm 2 sections and plated onto PARP selective media (contains pimarcin, ampicillin, rifampicin, and pentachloronitrobenzene). Phytophthora colonies emerging from the edge of baits were hyphal-tipped onto 20% clarified V8 juice agar (CV8A) to obtain pure cultures [1]. Cultures were maintained on CV8A and blocks of fresh agar cultures were transferred into microtubes with sterile distilled water for long-term storage at 15°C. The holotype isolate MYA-4946 was deposited at the American Type Culture Collection in Manassas, Virginia, USA.

Colony morphology and cardinal temperatures
Ten-day-old colony morphology of the four isolates of P. mississippiae on carrot agar (CA), CV8A, malt extract agar (MEA), and potato dextrose agar (PDA) grown at 20°C in the dark was noted and photographed.
Cardinal temperatures of the four isolates were assessed on CV8A and CA. Agar blocks (5 mm in diameter) taken from actively growing areas of 7-day-old cultures were placed on fresh media at the center of 10-cm Petri dishes. Triplicate Petri dishes per isolate per temperature were placed in the dark at 5, 10, 15, 20, 25, 30, 35, and 40°C. Two perpendicular measurements of each colony were taken after 8 days. This test was repeated. Following the analysis of variance using R statistical software v. 2.15.0 [28], data from repeating tests were pooled together. Radial growths along with their standard errors were plotted against temperature using the gplots package v. 2.11.0 [29] in R.

Morphology of sporangia and gametangia
Sporangia were produced by transferring agar plugs (10×10 mm 2 ) from actively growing area of 10-day-old cultures on CV8A to Petri dishes containing non-sterile, 1.5% soil water extract solution (SWE, 15 g of sandy loam soil/1 L distilled water) or filtered, non-sterile pond water. Mature sporangia developed after incubating at room temperature (c. 23°C) under cool-white fluorescent light.
Mating type of these isolates was determined by placing each with an A1 or A2 mating type tester of P. cinnamomi in dual cultures on hemp seed agar (HSA). Selfed sexual structures were produced at room temperature using the polycarbonate membrane method to physically separate P. mississippiae isolates from their reverse mating type testers [30,31]. Several heterothallic species including P. cinnamomi, P. Pythium aphanidermatum Pythium P1779 n/a n/a n/a GU983641 n/a P2 n/a n/a n/a n/a AY564163 T exo-type *n/a=not available

DNA extraction, amplification and sequencing
Isolates were grown in 20% V8 juice broth at room temperature for one week. Mycelial masses were harvested and lysed using a FastPrep ® -24 system (MP Biomedicals, Santa Ana, CA). DNA was extracted as instructed using the DNeasy ® Plant Mini kit (Qiagen, Valencia, CA). Amplifications were performed with forward primer ITS6 and reverse primer ITS4 [2] for the internal transcribed spacer (ITS) region covering ITS1, 5.8S rRNA gene, and ITS2, following previously described reaction mix recipe and PCR program [32]. Primer pair COX4FR was used to amplify the mitochondrial cytochrome c oxidase 1 (cox 1) gene [3]. Sequencing was performed in both directions at the University of Kentucky Advanced Genetic Technologies Center (Lexington, KY) using the same primers. Sequences of both directions were visualized with Finch TV v. 1.4.0. and aligned using Clustal W.

Sequence analyses
Sequences generated in this study were compared with those of all other species in the same clade and species representing other clades (Table 1). Sequences were aligned using Clustal W. Phylogeny reconstruction was conducted in MEGA 5.1 [33] using the Maximum Likelihood method based on the Tamura-Nei model [34] with 1,000 replications of bootstrap.

Colony morphology
The four isolates of P. mississippiae had a similar growth pattern at   CA was radiate with a smooth edge. Isolates 57J1, 57J3 and 57J4 had a moderate growth rate on CV8A while isolate 57J2 had a slow growth rate. All isolates produced hispid aerial mycelia. Colony pattern on CV8A was radiate to slightly petaloid with a relatively smooth edge. On MEA, all isolates had limited but discernible growth with irregular colony patterns. Isolates had a moderate growth rate on PDA and produced tomentose aerial mycelia. Colony pattern on PDA was petaloid (isolates 57J1, 57J2 and 57J3) to slight cottony (isolate 57J4).

Cardinal temperatures for vegetative growth
Radial growth rates were different among isolates (P<0.01) but not between repeating experiments (P=0.17). Isolates 57J1, 57J3, and 57J4 had similar growth rates (Figure 2A). Isolate 57J2 grew more slowly than the other isolates at 10 to 30°C on both CV8A and CA ( Figure 2B). The optimum temperature for the growth of P. mississippiae was 25°C on CA and 30°C on CV8A. Limited but notable growth was observed at 5 and 35°C. No growth occurred at 40°C. After the experiments were completed, cultures from all temperatures were relocated to room temperature. Additional growth occurred on plates previously maintained at 5°C but not those maintained at 40°C.

Sequence analyses and phylogenetic position
GenBank accession numbers of sequences generated in this study and used in the sequence analyses are shown in table 1. All isolates of P. mississippiae have 818 bp ITS sequences. Isolates 57J1 and 57J2 have an identical ITS sequence, while 57J3 and 57J4 have an identical ITS sequence (Table 2). These two subgroups differ by 3 bp. These ITS sequences of P. mississippiae were distinct from those of all known Phytophthora species. Two species with most similar ITS sequences are Isolate  ITS1  ITS2   15  43  44  98  106  173  469  517  553  582  588  608  652  740  744  787 Phytophthora mississippiae     Table 2).
The four P. mississippiae isolates have an identical 867 bp cox 1 sequence, which also is distinct from those of all known species. The cox 1 sequence of P. mississippiae differs from two proximal sequences, those of P. thermophila (GenBank accession no. HQ012872) and P. borealis (GenBank accession no. JQ626625) by 30 and 31 bp, respectively.
Sequence analyses of both ITS and cox 1 sequences placed P. mississippiae in clade 6 of the genus Phytophthora [5,9]. The four P. mississippiae isolates form a distinct taxon in the phylogenetic trees based on ITS ( Figure 3) and cox 1 (Figure 4)  Phytophthora mississippiae produced abundant sporangia in 1.5% SWE after 15 hours under light. Sporangia were mostly ovoid to obpyriform (Figures 5A-D, F-H, J). It occasionally produced slight ellipsoid sporangia (Figures 5E, I). Sporangia were noncaducous, mostly nonpapillate (Figures 5A-G) and sometimes semipapillate ( Figures 5H-J). Primary sporangia were terminal and averaged 60.5 µm in length and 31.7 µm in width. Secondary lateral sporangia were observed on the mycelial plug after submersion in SWE for more than 40 hours. Nested and extended internal proliferation was common ( Figures 5L, M). Sporangial characteristics among four P. mississippiae isolates are summarized in table 3. Mycelia were flat ( Figure 5N), coiled ( Figure 5O), or swollen ( Figure 5P). Hyphal swellings were commonly elongated with irregular shapes, especially in aged cultures (>30-dayold). Catenulate, globose hyphal swellings were frequently observed in both fresh and aged cultures ( Figure 5Q). Chlamydospores were not observed.
Phytophthora mississippiae is self-sterile. Gametangia were produced in dual cultures where P. mississippiae isolates were paired with an A2 mating type tester of P. cinnamomi suggesting that all four isolates examined in this study are A1. In the polycarbonate membrane test, gametangia were produced by isolates 57J3 and 57J4 after 50-day-breeding when paired with A2 mating type testers of P. cryptogea ( Figures 6A, B) and P. nicotianae ( Figure 6C, D). Oogonia had characteristic ornamented protuberances on the surface and oogonial wall was pigmented to golden-brown with maturation ( Figures 6A-D). Many oogonia had a tapered base ( Figures 6A, B

Habitat
Irrigation water of an ornamental plant nursery, Mississippi, USA.

Discussion
This study characterized a novel species of Phytophthora morphologically, physiologically and phylogenetically then named it as P. mississippiae. This is the first and critical step to understanding  the biology, ecology and economic significance of any novel species. The description of P. mississippiae will help the first responders in diagnosing the disease caused by this new species. It also will reduce misidentification of high-impact pathogens like P. ramorum [17] and P. kernoviae [35].
Like many other species in clade 6, the economic importance of P. mississippiae is not known at this point. Many clade 6 species are abundant in natural habitats and frequently recovered from natural water and soil environments, but usually do not cause apparent disease symptoms on plants [38]. Only a few clade 6 species have been found to cause diseases on agricultural and horticultural plants [5]. Examples include P. asparagi which causes root rot of asparagus [39] and P. megasperma which causes crown rot of hollyhock [8]. A saprophytic lifestyle for many species in this clade may play an important role in decomposing plant debris [38]. Unlike many other clade 6 species initially detected in natural environments, P. mississippiae was first recovered from irrigation water in a plant production facility. It is possible that P. mississippiae was carried into agricultural irrigation water systems from surrounding habitats through flash flood runoff that occurred in this area during heavy rains. This hypothesis is supported by the fact that this new species was found only in Mississippi but not in any of nursery irrigation systems surveyed in Virginia during the past 14 years and in Alabama during the past 2 years. Nevertheless, investigations into its origin, pathogenicity and host range are warranted.