Stimulation of sporangia and zoospore
Production of sporangia and zoospores is the principle means by which the numbers of infective units are increased and accounts for the rapid regeneration time of
Phytophthora species. Sporangia together with zoospores are the principal means of dispersal of the
Phytophthora spp. and as the most potent means ensuring penetration of the host (
Weste and Vithanage, 1979). In the present study, various methods given by earlier workers were attempted for induction of sporangia and zoospores of
P. cajani and results (Table 1) reveled that, none of the protocols are able to stimulate the sporangia and zoospores. Hence, the alternative method for induction of spornagia and zoospore was attempted. Taking clue from the study of culture media and mycelial growth of
P. cajani, tomato extract agar produced maximum growth therefore; further probed to develop a protocol for maximum production of sporangia and zoospores by diluting tomato extract broth with fresh water. It was found that diluted tomato extract broth could induce abundant sporangia and zoospores within 24 hours of incubation. Further same methodology could also induce the male and female gametangia
viz., antheridium and oogonium and sexual spore oospore within 48 hours. The induction of sporulation of
P. cajani using diluted tomato extract broth
in vitro is first of its kind. The induction of sporulation in tomato broth could be due to its precise availability of nutritional requirement for
Phytophthora in the medium as reported earlier by
Grant et al., 1984.
On observation found that the most important factor involved in the process of sporulation by
P. cajani is sudden reduction in food supply which forced the fungus to sporulation stage instead of the vegetative stage and stimulation of sporangial production thus initiating the cycle of spore formation. Major advantages of this protocol are easy handling, reproducible and rapid for obtaining the zoospores within 36 hours and enable us the inoculating large numbers of pigeon pea genotypes for resistance breeding against the Phytophthora blight disease. Any information relating to this zoospore is significant to understanding the pathogen and host × pathogen × environment interaction and ultimately to manage the disease.
Diplanetism
Diplanetism, the phenomenon observed in some members of the
Oomycetes, of there being two distinct motile phases, with morphologically different zoospores formed in each. In the study
P. cajani showed a diplanetism mechanism, in that one zoospore may germinate directly forming an emerging tube and producing secondary zoospores (Fig 3). The report of
P. cajani producing diplanetism mechanism is first of its kind. Similar report in
Phytophthora ramorum doneby
Moralejo and Descals (2011), where
P. ramorum displays monomorphic diplanetism and microcyclic sporulation, in that cysts may germinate directly forming a emerging tube or indirectly by releasing secondary zoospores. These secondary zoospores do not differ morphologically and can infect hosts. However, symptom expression is delayed and infectivity is lower than for primary zoospores. The diplanetism mechanism in the pathogen may provide a second opportunity for host infection and may increase the chance of dispersal of pathogen in the soil (
Moralejo and Descals, 2011).
Temperature and RH on number of zoospores using diluted tomato extract broth
Phytophthora has a complex asexual life cycle with distinct multiple infectious propagules which include the mycelium, sporangia and zoospore cysts (
de Souza et al., 2003). Each of these asexual developmental stages is crucial for plant infection and disease development (
Hardham, 2001). Different environmental factors such as temperature, water relations, physical and chemical conditions and interacting combinations of these factors have been reported to influence
Phytophthora pathogenesis (
Duniway, 1983). Moreover, the geographic distribution and seasonality of Phytophthora diseases are thought to depend on the constraints of any of these environmental factors on any or all of the life cycle stages (
Duniway, 1983). Of the environmental factors, temperature has been reported to have greatest influence on growth, reproduction and pathogenesis of
Phytophthora spp. (
Matheron and Matejka, 1992;
Timmer et al., 2000).
Studies on the influence of temperature and RH on sporangial production by
Phytophthora spp. is an important prerequisite for developing disease predictive models in the field. Hence, an attempt made to study the impact of temperature (20, 25, 30, 35 and 40°C) and RH (50, 75, 85, 95 and 100 %) on zoospore production using the diluted tomato extract broth. The results (Fig 4A) revealed that the greatest number being produced at 30°C (183 × 10
3 per ml), whereas few zoospores were produced at variation of 30°C
viz., 35°C (86 × 10
3 per ml) and 20°C (112 × 10
3 per ml), whereas at 40°C production of zoospore was nil. The results implied that the ambient temperature of 30°C has a more critical effect on reproduction of pathogen. Similar reports were made in other species of Phytophtohra
viz.,
P. citrophthora and
P. parasitica (
Matheron and Matejka, 1992);
P. infestans (
Mizubuti and Fry, 1998) and
P. palmivora (
href="#timmer_2000">Timmer et al., 2000).
Among all the RH levels, RH of 100 % induced highest number of zoospores (188 × 10
3 per ml) and statistically superior over others (Fig 4B). Similar observations has been reported in
Phytophthora pseudosyringae sp. nov
(Jung et al., 2003), P. infestance (
Minogue and Fry, 1981) and
P. capsici (
Granke and Hausbeck, 2010b) where RH played great role in influencing the amount of zoospore formation.
Zoospore concentration and plant infection
The optimum quantity of inoculum should be selected to characterize better relationship between inoculum concentration and infection
(Fraedrich et al., 1989; McIntyre and Taylor, 1976;
Milholland et al., 1994). Quantity of inoculum is bound to influence the disease incidence and infection takes place only when minimum inoculum potential of the pathogen is present in the soil.
Although, information is available on the numbers of zoospores required for infection of plants or plant parts on different species on
Phytophthora (
Halsall, 1977; Hickman, 1970;
Pratt et al., 1975), the information on the quantities of zoospores required to infect pigeonpea plants under flooded conditions is very limited. The study established the quantitative the relationship between numbers of zoospores of
P. cajani and infection in pigeonpea. The study indicated that, with the increase in concentration of zoospores there was substantial increase in the infection of plant. Of all concentrations, 1×10
-5 zoospores/ml induces maximum infection of plant (100 %) and shortest incubation period (24 hours), whereas only 6.6, 19.6 and 69.4 per cent infection was observed at 1×10
-2, 1×10
-3 and 1×10
-4 respectively (Fig 5). Further, incubation period is delayed as reduction in inoculum load. Shortest incubation period at higher concentration of zoospores caused due to initial infection by large numbers of zoospores and delayed incubation period is because of initial infection by low levels of zoospores. The results of this study are in general agreement with work with
P. parasitica var.
nicotianae on tobacco (
Gooding and Lucas, 1959),
P. palmivora on papaya (
Ko and Chan, 1974). The amount of initial inoculum determines the amount of disease. The disease increased with the increase in an amount of inoculum concentration as availability of infection courts and at certain stage addition of extra inoculum did not increase the amount of disease. This could be due to saturation of all the infection courts
Van der Plank (1975).
Symptomology of disease
During the periodical survey at research farm, ICRISAT the symptoms were recorded and presented in Fig 6. Infected young seedlings showed crown rot symptoms soon after their emergence, later collapsed and died within 4 days. In older plants,
i.e. on 1 to 2 month old plants, water soaked lesions of varied size and shape appeared on the primary and trifoliate leaves (Fig 6a). As the disease progressed, the entire foliage blighted (Fig 6b). Brown, dark brown or black lesions appeared on the collar region above the ground level on the main stem and branches (Fig 6c; 6d). During severe infection, the lesions increased in size and encircled the stems causing them to shrink. The stem above the lesion girdled dried out and broke at the infection site (Fig 6e). In late infections, lesions on stems developed into cankers or galls and the infected bark cracked (Fig 6f).
Williams et al., (1975); Nene et al., (1979); Vishwa Dhar et al., (2005) and
Pande et al., (2011) observed similar type of symptoms in the field and under greenhouse conditions.
