Morphological characterization
Detailed recognition of features of mycelia and characteristics of asexual and sexual structures is very important for correct understanding of the whole infectious process and disease development. The features of the sporangium, like shape, type of a papilla, caducous or deciduous nature of sporangia and subsequent length of the subtending pedicel are important key features for identification of some species of Phytophthora (
Erwin and Ribeiro, 1996). In the foregoing study, an effort was made document the various micro morphological characters of pathogen such as hyphae and hyphal swelling, sporangial morphology, gametangial morphology, chlamydospores and colony characters (Table 1 and Fig 1).
Micro morphology
Hyphae and hyphal swelling
Mycelium was hyaline, branched, coenocytic filamentous measuring average breadth of 4.07 µm. Hyphal swelling was very common and it was both terminal and/or intercalary with lengths × breadth of 17.9 × 13 µm. The results are in corroboration with other member species of clade 7 group of Phytophthora
viz.,
P. vignae (
Gallegly and Hong, 2008) and
P. cinnamomi (Robin et al., 2012) where hyphal swellings are abundant and positioned intercalary and rarely terminal.
Sporangial morphology
Size and shape of sporangia of
Phytophthora vary due to culture media and environmental conditions (
Waterhouse 1963). Size and shape differences among sporangia and oospores can contribute to easier identification of
Phytophthora species from infected plant tissues
(Hardham et al., 1994; Hardham, 1995).
Kaosiri et al., 1978 reported that, caducity of sporangia is a useful taxonomic tool in identification of many
Phytophthora species.
In the study sporangiaphore produced by
P. cajani were simple or sympodial and average breadth is about 3.6 µm. These observations were in accordance with
P. cambivora (
Wicks and Lee, 1986)
, P. melonis (
Ho et al., 1995),
P. cinnamomi (
Robin et al., 2012) and
P. sojae (
Faris et al., 1989) where they exhibit simple or sympodial branching and typically proliferate through the empty sporangium.
Sporangium was oviod-obpyriform with an LxB ratio of 1.56 µm; hence it was an oviod-obpyriform. Sporangia were of proliferating type which emerged externally from the base of previous sporangium and produced a new sporangium. Further sporangia were nonpapillate and noncaducous, where sporangia did not detach at maturity. Sporangial exit pore is very narrow with 7.3 µm breadth. Released zoospores were encysted with diameter of 11.7 µm.
Sporangial characteristics of
P. cajani were in accordance with other species of clade-7 phylogenetic group of
Phytophthora where
P. cambivora (
Wicks and Lee, 1986) and
P. alni (
Brasier et al., 2004) exhibits the oviod-obpyriform sporangium with L:B ratio of 1.6 µm and 1.32-1.62 µm respectively.
Phytophthora cinnamomi (
Robin et al., 2012),
P. alni (
Brasier et al., 2004;
Cerny et al., 2008), P. cambivora (Vannini and Vettraino, 2011),
P. sojae (
Kaufmann and Gerdemann 1958)
, P. fragariae (
Ho and Jong, 1988),
P. vignae (
Purss, 1957) where all the species were producing nonpapillate and noncaducous sporangia.
Gametangial morphology
The mating behaviour of
Phytophthora species is very important especially in relation to the survival of the species.
P. cajani was homothallic and produced the male and female gametangia called oogonium and antheridium. Amphigynous type of sexual reproduction was observed where both male and female gametes orient perpendicular to themselves and produced a sexual spore called oospore. Average diameter of mature oogonia is about 23.1 μm whereas oogonium stalks length measures about 7.8 μm. Length and breadth of antheridium is of 6.6 × 9.3 μm. Average diameters of oospores is about 18.3 μm.
Phytophthora cajani differs from some other of species of clade 7 group
viz.,
P. cambivora (Heffer
Link et al., 2002), P. melonis (
Ho, 1986)
, P. cinnamomi (Robin et al., 2012) by being homothallic. Further
P. fragariae (
Ho and Jong, 1988) and
P. sojae (
Faris et al., 1989) were though homothallic but differs with
P. cajani in antheridial configuration as they were either paragynous or amphigynous.
No chlamydospores were produced by test pathogen in any of the applied methods. Similar observation was observed in
P. inundata (Brasier et al., 2003b), P. alni (Ho et al., 1984), P. fragariae (
Milholland, 1994) and
P. sojae (
Kaufmann and Gerdemann 1958) where chlamydospores were not produced
in vitro. In contradiction to
P. cajani other phylogenetically related clade 7 group species
P. cinnamom (
Robin et al., 2012) and
P. vignae (
Purss, 1957) were able to produce chlamydospores
in vitro.
Macro morphology
Colony characteristics and growth rates were useful as a first step in identification of
Phytophthora species. Biological features and cultural characteristics of genus
Phytophthora are very important for simpler identification (
Cahill and Hardham, 1994).
Colony pattern
Colony patterns are dependent on both culture media and isolate and show great variability.
Phytophthora spp. has a distinctive set of morphological traits that can be observed in culture on certain media (
Erwin and Ribeiro, 1996).
P. cajani colonies on tomato extract agar medium was characterised by dull white in colour, flat and rosette pattern type. The colony characteristics of
P. cajani varied among the culture media from dull white to cottony white colour growth which was flat to aerial with smooth to irregular margins (Table 2). The results were in accordance with other researchers on different culture media on colony pattern of
Phytophthora spp. (
Erwin and Ribeiro, 1996). Variability of colony morphology on different culture media is common throughout the genus of
Phytophthora. Thus, the usefulness of colony morphology as an identification aid beyond a supplementary purpose is questionable (
Erwin and Ribeiro, 1996;
Widmer, 2009).
Radial growth
The radial growth and sporulation of
P. cajani was studied on different media (Table 2). After growing at 30
oC for six days
P. cajani showed the fastest growth on tomato extract agar medium (90 mm) and next best was V8 juice agar medium (89.0 mm) and least growth of 40.67 mm was observed on carrot agar medium. However sporulation was not observed in any of the medium tested. The results were in accordance with
Ribeiro (1977), who reported that V8 juice agar as the best medium for the growth of many
Phytophthora spp. Similarly,
Dhingra and Sinclar (1995), stated tomato extract agar medium was ideal for growth and sporulation of
Phytophthora spp. Lack of sporulation in
Phytophthora spp. was probably due to a failure to meet some precise requirement mineral nutrition and temperature for this process to occur
(Grant et al., 1984).
Physiological characterization
Temperature-mycelia growth relationships
Temperature is one of the pre-requisite for the growth and sporulation of the fungus which plays an important role in infection and disease development. The growth of
P. cajani was tested on tomato extract agar medium at different temperatures of 5, 10, 15, 20, 25, 30, 35 and 40
oC and the results are summarized in Table 2. The fastest growth was observed at 30
oC (90.0 mm) followed by 25
oC (87.4 mm). Pathogen did not show any growth when plates were incubated at temperature of 5, 10 and 40
oC whereas 35
oC showed the growth of 23.5 mm hence
P. cajani can tolerate high temperature of 35
oC. Sporulation was not observed in all the temperatures tested. The result fastest mycelia growth at 30
oC concur with the findings of
Pal and Grewal (1975);
Kannaiyan et al., (1980) and
Mishra et al., (2010) who reported maximum vegetative growth of
P. cajani at 30
oC.
Relative humidity (RH) - mycelia growth relationships
Relative humidity is another important epidemiological factor for influencing physiology of fungal growth and sporulation.
P. cajani was growin at 30oC for six days at different RH. The result implied that, fungus prefers RH of 75 to 100% as evidenced by mycelial growth of 100 mm as against 82 mm in 50 and 55 % RH (Table 2). Sporulation of pathogen was absent at all the RH tested throughout the experiment. The results are in accordance with
Phytophthora capsici (
Granke, 2011) and
P. parasitica (Prasad et al., 2017) where faster growth of the fungus was observed by increasing the RH.
