Means of cultivation

The culture media selected to isolate, describe and identify Phytophthora species are of paramount importance. One factor that must be considered in choosing the appropriate medium is the Phytophthora species with which it is being worked (^{Erwin and Ribeiro, 1996}). The selective means to isolate Phytophthora contain fungicides and antibiotics, to avoid the growth of fungi and bacteria.

The most commonly used selective media are PARPH, PARP, PARPH, PVP (P-pimaricina, A-ampicilina, R-rifampicina, P-pentacloronitrobenceno, H-himexazol, V-vancomicina), 3-P (P-pimaricina, P-Penicinila, P-Polimixina B, CARP (C-cornmeal agar, A-ampicilina, R-rifamicina, P-pimaricina), CARPD (D-dicloran), CARP+ (+-benlato e himexazol), there are variations in the concentrations of the antibiotics and fungicides are added (^{Tsao and Ocana, 1969}; ^{Jeffers and Martin, 1986}; ^{Erwin and Ribeiro, 1996}; ^{Drenth and Sendall, 2001}) these must be added after the medium has been sterilized the medium. Pimaricin is an expensive antibiotic for laboratories in Latin America; however, it can be replaced by Delvocid Instant (^{Erwin and Ribeiro, 1996}), which is inexpensive, it contains Natamycin, which is why this medium is known as NARPH. Likewise, the himexazole that previously came from Japan, is currently available in different countries. The selective medium can be prepared using corn flour agar or V8 agar. When corn flour can not be purchased, it can be replaced by corn starch (Felix- Gastelum. Comun. Pers., 2015).

Some examples in which the medium is quite specific is that of rye agar, which is mainly used to isolate P. infestans, P. ipomoeae and P. mirabilis, 3-P media and PCH (P-pimaricina, C-cloranfenicol, H-himexazol) are specific for isolating P. cinnamomi from roots and soil (^{Drenth and Sendall, 2001}; ^{Ivors, 2016}). The protocols and media (V8 agar, liquid V8, super V8, V8-rye, rye A, rye B, oatmeal, 5% carrot agar, fresh lime and sucrose), currently employed are described in the ^{Ivors (2016)}. In each laboratory, culture media can be prepared with the components available, it is also recommended to test different methods of preparation and select the most suitable ones for the individual needs.

Direct diagnosis

When a phytopathological diagnosis is carried out, the results are expected to be obtained as quickly as possible, for which kits, ELISA tests, fluorescence in situ hybridization (FISH), hyperspectral techniques, antibody based biosensors, immunoflorescence, fluorescence, thermography, and test strips for the direct detection of pathogens in the field that are of great help and can detect Phytophthora (^{Miller et al., 1994}; ^{Fang and Ramasamy, 2015}).

Most field-use kits based on serological techniques give a rapid on-site response in a few minutes or hours, which is of great use to producers, consultants, technicians and inspectors. Tests can be performed from any plant tissue that is required to be analyzed and the result is easy to interpret. If necessary, the results can be confirmed later in the laboratory. Some of the kits available on the market are Phytophthora late blight Pocket Diagnostic^® test for the detection of P. infestans in potato, Phytophthora spp. ALERT-LF^® and ImmunoStrips to identify infections caused by Phytophthora in a wide range of crops including nursery plants. These kits are designed for use in field or laboratory from plant tissue samples, do not require special equipment to carry them out, since all the required material is included.

Isolation of Phytophthora

Isolation of Phytophthora from plant tissue. A suitable collection and selection of samples determine the success in the process of isolation of Phytophthora from diseased plant tissue. It is recommended to collect samples of leaves, stems, roots, fruits, flowers, etc, that have a zone of healthy tissue in front of the advancement of the lesion(Figure 1). To isolate sections of tissue from the area mentioned should be cut, as it will be where the pathogen is in active growth. These tissues can be planted without disinfesting, directly in a selective medium (^{Erwin and Ribeiro, 1996}; ^{Streito, 2002}; ^{Jung and Blaschke, 2004}; ^{Jung and Burgess, 2009}; ^{Kasuga et al., 2012}).

Figure 1 Isolation of Phytophthora from roots with rot in selective medium NARPH-corn meal. 

It is not recommended to isolate Phytophthora from necrotic tissue due to the presence of fungal and saprophytic bacteria. Some authors suggest to disinfect the tissues superficially, for which the sections of tissue are immersed in 70% ethanol for 10 to 30 s, then washed with sterile distilled water, then dried with sterile filter paper and finally placed in selective culture medium (Hüberli, 2000; ^{Davison and Tay, 2005}; ^{Ghimire et al., 2009}; ^{Moralejo et al., 2009}; ^{Tsopmbeng et al., 2012}), in some reports it is mentioned that alcohol does not leave residues so it is not necessary to rinse (^{Martin et al., 2012}; ^{Borines et al., 2014}).

A 10% commercial chlorine solution (5% sodium hypochlorite) is also used mainly. The disinfestation process involves immersing the plant tissue in the chlorine solution for 30 s, then rinsing with sterile distilled water and then drying with sterile paper, finally cutting the tissue sections before placing them in the selective medium (^{Drenth and Sendall, 2001}; ^{Ghimire et al., 2009}).

The disinfestation time may vary, which depends on the type of tissue from which the insulation is to be performed. It is important to take into account that sensitivity to chlorine varies from one species to another of Phytophthora. ^{Hong et al., (2003)}, have shown that the sensitivity to this is pathogen-dependent. Some species of Phytophthora may be sensitive to chlorine and ethanol and should be omitted (^{Hong et al., 2003}). When it is unknown if the species is sensitive to these compounds, it is recommended to disinfect only some sections of tissue with ethanol or chlorine and others to seed directly in selective medium.

The tissue should preferably be dried with filter paper or sterile paper towels before being sown in any medium to prevent bacterial growth (^{Erwin and Ribeiro, 1996}). Tissue sections seeded in selective medium should be incubated at a temperature favoring Phytophthora growth (^{Erwin and Ribeiro, 1996}; ^{Ferguson and Jeffers, 1999}; ^{Drenth and Sendall, 2001}; ^{Martin et al., 2012}).

Isolation of Phytophthora of water. Irrigation water is an important source of inoculum of plant pathogens. The genera Phytophthora and Pythium of the family Pythiaceae are the most common, destructive and polluting in irrigation systems (^{Erwin and Ribeiro, 1996}; ^{Drenth and Sendall, 2001}; ^{Loyd et al., 2014}; ^{Zappia et al., 2014}). More than 30 species of Phytophthora have been detected in water in the world, with some species not yet tested for pathogenicity (^{Zappia et al., 2014}), others that have been inoculated in different hosts have not been pathogenic (^{Copes et al., 2015}).

Only the pathogenicity of some P. irrigata in azalea, chili and tomato has been demonstrated; P. hydropathica in azalea, cineraria and tomato; P. megasperm in azalea and chili; P. nicotianae in vinca, begonia, eggplant, chili, cineraria, lupinus and tomato; P. palmivora in azalea, begonia, eggplant, chilli and tomato; P. tropicalis on azalea, begonia, eggplant, chili, cineraria, lupinus, tomato and vinca (^{Hong et al., 2008}; ^{Loyd et al., 2014}). Water reservoirs or channels for agricultural use can be contaminated by water runoff from fields infested with Phytophthora, which may also lead to the dispersion of the pathogen to places where it was not present (^{Erwin and Ribeiro, 1996}).

Recirculated water infested within and between nurseries favors the dispersal of Phytophthora propagules in the form of zoospores, healthy plants and areas where it was not found (^{MacDonald et al., 1994}; ^{Stewart-Wade, 2011}). Therefore, effective protocols to detect this oomycete in irrigation water are essential. Filtration methods can process limited amounts of water compared to the enormous amount of water that exists in dams, bodies of water and irrigation systems.

In a study by ^{Hong et al. (2002)}, obtained higher recovery rates in different water sources by filtering the water samples through a membrane that retains the propagules, which is then inverted and placed directly in the selective PARP-V8 medium. The membranes tested were of different materials and pore sizes, observing better results with the Millipore 5 and Durapore 5 membranes (Millipore^®) obtaining higher recovery rates.

Isolation of soil Phytophthora or planting substrate

Phytophthora can be dispersed to new sites through the sale of different seed substrates or propagule-infested soils (^{Ferguson and Jeffers, 1999}; ^{Dart et al., 2007}; ^{Dart and Chastagner, 2007}). It is recommended to use different trap vegetable tissues with each soil sample or substrates suspected to be contaminated with Phytophthora to rule out false negative results (^{Jeffers and Aldwinkle, 1987}; ^{Davison and Tay, 2005}b; ^{Balci et al., 2007}; ^{Tooley and Carras, 2011}). A wide range of trap plant tissues are used to recover Phytophthora from soil since not all are colonized in the same way by different Phytophthora species (^{Erwin and Ribeiro, 1996}). In a later paragraph the recommended trap vegetable tissues are mentioned.

To detect Phytophthora in soils or substrates, flooding of these with sterile distilled water is used, in such a way that propagules, especially zoospores are released into the water, swim to the surface and infect the plant tissue used as trap plant tissue (Figure 2). Some proportions used to mix substrate and water are 1:2, 2:3.5 (weight: volume). This method has several advantages over tissue insulation, since large amounts of soil can be analyzed, increasing the chances of detecting the pathogen when the inoculum concentration is low.

Figure 2 Rhododendron leaves used as plant tissue trap to detect the presence of Phytophthora in substrate or soil samples, in Petri dishes. 

It is also more feasible to detect oospores in homotactic species (^{Erwin and Ribeiro, 1996}; ^{Balci et al., 2007}; ^{Fichtner et al., 2007}). Zoospore-colonized trap tissues are subsequently washed with tap water or sterile distilled water to remove bacteria from the tissue surface, dried with sterile paper and seeded in selective medium (^{Ferguson and Jeffers, 1999}; ^{Jung et al., 2000}; ^{Balci et al., 2007}; ^{Fitchtner et al., 2007}; ^{Hwang et al., 2008}; ^{Ghimire et al., 2009}). The petiole of Rhododendron leaves is the tissue that is generally infected first, so that it can be cut and planted in selective medium (Figure 3).

Figure 3 Necrotized petioles sectioned from Rhododendron leaves, previously placed with soil or planting substrate and transferred to selective médium NARPH prepared with V-8 agar. 

The collection of samples for purposes of detection or population density should be performed in the different seasons of the year since populations may vary from one station to another (^{Erwin and Ribeiro, 1996}). Most species of Phytophthora are favored by conditions of high humidity, temperatures between 15 and 35 °C, humid climates with abundant rains and nutrient-poor substrate or soil. When these conditions are present, it is advisable to analyze samples of the substrate or soil to detect the pathogen (^{Erwin and Ribeiro, 1996}; ^{Drenth and Sendall, 2001}; ^{Kong et al., 2009}).

It is suggested that the collections are made from moist soil, near apparently healthy roots and from those that are growing (^{Erwin and Ribeiro, 1996}; ^{Balci et al., 2007}; ^{Fichtner et al., 2007}). Samples should not be exposed to temperatures above 45 °C or to freezing temperatures to maintain the viability of the pathogen (^{Drenth and Sendall, 2001}). They should be placed in plastic bags to prevent dehydration and deposit in a cooler for transportation to the laboratory; placing an insulation material to avoid direct contact of the samples with the ice. It is recommended to keep them moist, at a temperature between 10 and 20 °C and should be processed as soon as possible (^{Drenth and Sendall, 2001}).

Vegetable trap tissues

For the detection of Phytophthora to be more effective from soil and water it is recommended the use of vegetal tissues trap. Selection of tissue type is important for the recovery of Phytophthora species. The trap vegetable tissues that are used can be of different plants and different plant parts such as: cotyledons, small leaf discs, fruits, complete leaves, petals, seedlings, seeds and seed pods. The choice of plant tissue trap depends on the species of Phytophthora suspected to be the causal agent of the disease. The leaves of Rhododendron, Camellia, Quercus, Lupinus, Juniperus needles, apple, pear and cucumber fruits are the most used by which a large number of Phytophthora species can be detected (^{Erwin and Ribeiro, 1996}; ^{Ferguson and Jeffers, 1999}; ^{Ivors et al., 2004}; ^{Balci et al., 2007}; ^{Gevens et al., 2007}; ^{Sutton et al., 2009}; Reeser et al., 2011; ^{Tooley and Carras, 2011}; ^{Themann et al., 2002}; ^{Huai et al., 2013}). A greater number of Phytophthora species are recovered when whole leaves of the plant tissue trap are used (^{Ferguson and Jeffers, 1999}; ^{Ghimire et al., 2009}), than when discs of leaves are used, since it is not recommended because they are easily colonized for Pythium and bacteria.

Purification of bacteria contaminated isolates

To make storage of the strains and studies of morphological and genetic characterization of Phytophthora it is a requirement to obtain isolates free of bacterial contamination. Isolates that are obtained in media with antibiotics are sometimes not free of bacteria. It is recommended to add more than one antibiotic to the medium used to make the isolates (^{Erwin and Ribeiro, 1996}; ^{Nesbitt et al., 1981}) to inhibit the growth of different types of bacteria. On the other hand the use of antibiotics can induce resistance in the bacteria and its use in high concentrations inhibits the germination of Phytophthora spores (^{Erwin and Ribeiro, 1996}).

Some alternative methods to the use of antibiotics and low cost are the method of overlapping and the use of acidified medium (^{Solache-Huacuz et al., 2010}; ^{Martin et al., 2012}). The first consists of placing a cube of medium with contaminated mycelium, with mycelial growth upwards, in an empty sterile Petri dish, on the cube a triangle of sterile medium is placed (Figure 4).

Figure 4 Purification of a Phytophthora isolate contaminated with bacteria, a piece of contaminated mycelium can be placed under a triangle of sterile medium. 

The mycelium will grow through the medium triangle towards the bacteria-free surface. The second consists of acidifying the culture medium to pH 3, using 0.14% tartaric acid (^{Koike, 2007}; ^{Solache-Huacuz et al., 2010}) or PDA (potato dextrose agar) at pH 4 with 25% lactic acid (^{Erwin y Ribeiro, 1996}). When the mycelium begins to grow, a test must be performed to confirm its purity in Luria Bertani liquid medium (LB) in test tubes, or if there is some other medium that favors the growth of bacteria can also be used. Medium cubes are cut with mycelium, placed inside tubes and incubated at room temperature for 24-48 h (^{Erwin and Ribeiro, 1996}; ^{Solache-Huacuz et al., 2010}). Before storing the isolates or performing some study it is necessary to carry out this test, to ensure that they are pure (Figure 5).

Figure 5 Appearance of a Phytophthora colony of a pure culture, the mycelium does not present color since it does not have pigments. The growth pattern of the colony may vary. 

Morphological identification

Species identification is performed with the support of tabular cues (^{Waterhouse et al., 1963}; ^{Martin et al., 2012}). For this, the cultures to be identified must be pure, that is to say obtained by zoospores or hyphal tip. The genus Phytophthora, is considered difficult to identify especially for those who have no experience (^{Waterhouse et al., 1963}), since the morphological differences between some species of Phytophthora are small and the morphological characters are influenced by the environment, and in some species such as P. capsici present sporangia with different forms. Also, the increasing number of species described, that some species of Phytophthora do not produce sporangia in the media that are commonly used, and the natural presence of hybrids between species of Phytophthora (^{Man in ´t Veld et al., 2007}), complicates the morphological identification.

The production of sporangia can be induced in several ways: a) by adding sterile distilled water to disks or blocks of V-8 medium with mycelium; b) adding an unsterilized soil extract suspension; and c) adding saline to the blocks (^{Eye et al., 1978}; ^{Schoulties et al., 1980}; ^{Fichtner et al., 2012}; ^{Almaraz-Sanchez et al., 2013}).The choice of liquid to be added will depend on the species of Phytophthora being studied. For example, P. cinnamomi suggests using non-sterile soil extract, incubating the isolates under fluorescent white light and performing fluid changes until sporangia are observed (^{Chen and Zentmyer, 1970}; ^{Eye et al., 1978}; ^{Ahumada et al., 2013}).

Rainwater can also be used to induce sporulation. To perform species identification, there is currently a tabular key published by ^{Martin et al. (2012)} and a lucida key available on the internet that includes most morphological features and species (http://idtools.org:8080/key-server/player.jsp?keyId=48). For identification of species, these keys take into account characteristics of asexual spores: sporangium type and shape, dimensions (length and width), number of papillae, expiration, pedicel length, base shape and some sporangiophore characteristics as type of branching, as for sexual spores, type of anteridium, characteristics and dimensions of oogonium and oospora, some additional characteristics for the identification of species of the genus Phytophthora are the presence or absence of chlamydospores, temperature range at which they grow optimal growth temperature, presence or not of swelling in the mycelium and type of reproduction of the strain (homotallic or heterothal) (^{Martin et al., 2012}).

Molecular identification

Modern molecular techniques based on the sequencing of specific regions of DNA are useful tools for the identification of Phytophthora species, since they are an indispensable complement of morphological characterization. These techniques are necessary because of how complicated it can be to determine the morphological characteristics of Phytophthora isolates, for the reasons mentioned above. However, some laboratories do not have the resources for equipment and materials needed to carry out this type of study, so it remains a limitation in many developing countries.

The locus with the highest number of sequences in the databases including all the Phytophthora species described, is the region of the internal transcribed spacers (ITS). The oligonucleotides used primarily for the amplification of the ITS region are ITS4 and ITS6 (^{White et al., 1990}; ^{Cooke et al., 2000}; ^{Martin et al., 2012} ). However, the sequences obtained from this locus do not always allow detecting interspecific variation in species closely related phylogenetically, so it is advisable for a correct identification to sequence several nuclear loci. Other commonly used loci are nuclear β-tubulin, and mitochondrial cytochrome c oxidase (CO1, cox2) (^{Bilodeau et al., 2007}; ^{Blair et al., 2008}; ^{Robideau et al., 2011}). In addition, the real-time PCR technique has provided results that allow the detection and quantification of the pathogen directly in the host (Lees et al., 2012, Engelbrecht et al., 2013).

The development of new technologies in mass sequencing of nucleic acids as metagenomics, allow the detection and discovery of plant pathogens. The new and third generation sequences are relatively new technologies of massive nucleic acid sequencing very useful for the diagnosis and identification of Phytophthora species. Which create profiles of large amounts of sequences allowing to reveal each and every one of the organisms present in the sample (^{Espindola et al., 2015}). These technologies are becoming less expensive, which will allow species identification at the genome level in the near future.

The databases that are considered reliable for identification of Phytophthora isolates through sequences can be found at the following links: http://www.phytophthoradb.org, http://www.phytophthora-id.org and http://www.q-bank.eu, since they have confirmed sequences with which they can be compared by BLAST analysis (^{Martin et al., 2012}).

Conclusions

Morphological and molecular identification is complemented, particularly when new species are found. Using only molecular techniques allows finding a greater number of species; however, it is not possible to characterize them morphologically, which is a limitation for their description. Also, timely diagnosis and correct identification of Phytophthora species are essential to prevent the spread of the pathogen, which will contribute to the proper management of the diseases they cause.