Research Article

Morphological Characterization of Phytophthora infestans and its Growth on Different Growth Media

Waqas Raza1*, Muhammad Usman Ghazanfar1, Muhammad Asif2, Ikram-ul-Haq3, Muhammad Zakria4 and Laith Khalil Tawfeeq Al-Ani5,6

1Department of Plant Pathology, College of Agriculture, University of Sargodha, Pakistan; 2Department of Agronomy, College of Agriculture, University of Sargodha, Pakistan; 3Department of Plant Breeding and Genetics, College of Agriculture, University of Sargodha; 4Crop Diseases Research Institute, National Agricultural Research Centre (NARC), Islamabad; 5Department of Plant Protection, College of Agriculture, University of Baghdad, Iraq; 6School of Biology Science, University Sains Malaysia, Minden, Pulau Pinang, Malaysia.

Received | January 24, 2022; Accepted | June 12, 2022; Published | September 15, 2022

*Correspondence | Waqas Raza, Department of Plant Pathology, College of Agriculture, University of Sargodha, 40100, Pakistan; Email: [email protected]

Citation | Raza, W., M.U. Ghazanfar, M. Asif, I. Ul-Haq, M. Zakria and L.K.T. Al-Ani. 2022. Morphological characterization of Phytophthora infestans and its growth on different growth media. Sarhad Journal of Agriculture, 38(4): 1189-1202.

DOI | https://dx.doi.org/10.17582/journal.sja/2022/38.4.1189.1202

Keywords | Sporangia, Carrot agar, Rye agar, Corn meal agar, Phytophthora infestans

Copyright: 2022 by the authors. Licensee ResearchersLinks Ltd, England, UK.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

Introduction

Potato (Solanum tuberosum L.) is third significant crop followed by rice and wheat and rice which consumed by more than billions of people worldwide (Birch et al., 2012; Arora et al., 2014; Mahr, 2021). Several factors both of plant pathogens and pests are causing the lower in yield and tuber quality (Kiptoo et al., 2021; Mahr, 2021; Nisa et al., 2022). The plant pathogen P. infestans (Mont.) de Bary causes very interesting disease called potato late blight that is considered most significant disease economically (Cooke et al., 2011; Zang et al., 2017). The Phytophthora genus was observed for the first time in 1876, and by 2012 had about 117 described pathogen’s species (Martin et al., 2012). P. infestans was identified as Botrytis infestans (Montagne, 1845) previously, and then Peronospora infestans (Mont.) Casp. (Caspary, 1879) had distinctive conidiophore characteristics, which he observed diverse enough being assigned as new genus name. Recently, Phytophthora spps. were considered among the organisms like fungi group because they have many morphological characteristics same, such as filamentous hyphal growth, asexual reproduction, and nutrition by absorption (Adl et al., 2005; Fry, 2008; Jaimasit and Prakob, 2011).

The late blight symptoms are small spots, typical V shaped, circular to irregular water-soaked at margins of leaves. Under most favorable environmental conditions the spots can expand quickly and white mycelia growth is observed during wet and humid conditions on the lower surface of the leaves (Flier et al., 2001; Fry, 2008). The survival of pathogen is having the ability to high sporulation and dispersed by wind and water. The epidemic progress is robustly dependent on optimum temperature range between 15-25°C and percentage relative humidity (100%) during pathogen’s life cycle (Ho, 2018). Meanwhile, higher temperature, above 30°C, would be responsible to stop the pathogen growth but it can start to sporulate again with respect to favorable conditions (Ribeiro et al., 2013). The asexual reproduction of P. infestans is by producing sporangia and zoospores while oospores in the sexual reproduction (Tyler, 2002). The exchange of genetic material through three mechanisms might be involved in the phenotypic character of P. infestans including sensitivity to fungicide, aggressiveness, ability of sporulation, latent period, morphological change, etc.

However, the phenotypic distinction in the population of P. infestans needs further investigations in the wide world. Changes in the P. infestans population in the early 1980s (Gotoh et al., 2005), as well as frequent physiological studies of this pathogen across the world, have been documented by many researchers (Jaimasit and Prakob, 2011). The characterizations of macro-morphology include the variation in colony type and growth rate but micromorphology comprised the change in the size of the sporangium, and zoospores (Bower et al., 2007). Micromorphology have been used as a varying factor for P. infestans while the investigation of morphological, cultural and physiological variability within pathogen population is the most helpful in understanding host-pathogen interaction, epidemiology and developing strategies for disease management (Bower et al., 2007).

Various synthetic media and different substrates include sweet corn, pea seeds, rye seeds, carrot media, bean meal, field corn, oatmeal, chickpea, V8 juice, cereal grains, and lima bean (Caten and Jinks, 1968; Goth, 1981; Peters et al., 1998; Medina and Platt, 1999; Sanyong et al., 1993). That is helpful in developing the sporulation and long-term storage of P. infestans (Erwin and Ribeiro, 1996) because pathogen is hard to isolate and purify on general media. Among all media Rye Agar media is the most frequently available organic substrate based medium (Hartman and Huang, 1995) but it is not commonly available in Pakistan and is precious to import. Knowledge of variability in morphological, cultural parameters of P. infestans is still scarce. Therefore, the current study is aimed at exhibiting the morphological characteristics and to determine the growth and sporangia production on different media of the pathogen P. infestans which causes potato late blight.

Materials and Methods

The research work was planned in the Departmental laboratory of Plant Pathology, University of Sargodha during 2017-18 to determine morphological characteristics of the pathogen (P. infestans) isolates collected from different potato growing areas of the Punjab, Pakistan.

Sample collection

Survey for assessment of late blight was conducted in the major growing areas of potato (Figure 1) in Punjab viz. Khushab, Okara, Jhang, Sargodha, Sahiwal, and Chiniot during the cropping seasons of 2017 and 2018. Each district was represented by three locations and each location was further divided into three sites. Five plants were randomly selected from four corners and centre of the plot representing each site. Diseased samples were collected in plastic polythene bags from the fields and brought to laboratory for further processing.

The infected leaf samples took during the occurrence of late blight from potato growing areas of Punjab. Each sample was labeled with the place, date of collection. The single lesion leaves with the most obvious symptoms (white mildew was visible) selected. The lesion was cut into small pieces (2 - 5 mm2) at the ad

 

vancing margin of the lesion and then inoculated onpetri plates containing Rye agar media as well as potato tubers for sporulation. The Petri plates were then incubated at 17-18ºC. Mycelia grew of samples for all isolates between 4-7 days depending on the virulence. The mycelia were transferred aseptically onto specific media plates for isolating purification in the next step.

Isolation and purification of Phytophthora infestans

The white colony from tuber from inoculated plates was removed and inoculated on plate medium antibiotics (rifampicin 20 mg. mL-1 or ampicillin 200 mg. mL-1) and stored for 8-10 days at 18-20ºC. The pure colony of P. infestans was transferred to slant further for long-term storage at 10-12ºC and maintenance as described by Raza and Ghazanfar, 2019. Pure cultures were observed to maintain for periods of up to six months on 9 cm Petri plates (25 ml agar per plate) using Rye agar A media (Young, 2007).

Pathogenicity test

Pathogenicity test was carried out to confirm the pathogen, P. infestans. For this purpose, healthy potato leaves were detached from six weeks old plants grown in green house, washed with distilled water for ≈10 minutes, and air dried to remove moisture. Leaves were covered to reduce leaf desiccation with pieces of moist cotton. The inoculated leaflets were then placed adaxial side up into moist box placed in incubator at 16-18°C in the dark for 4 days with 14 h illumination and 10 h dark photoperiod respectively (Figure 2) as described (Flier et al., 2007). The sporangial suspension was produced from 7 day fresh cultures by lightly washing the mycelium with distilled water and adjusted to about 60,000 sporangia/ml with the help of haemacytometer (Pliakhnevich and Ivaniuk, 2008). The spore suspensions were also produced from lesions which were placed into polypropylene culture tubes (14 ml) with 3-4 ml of preservative solution (0.2 M sodium acetate, acetic acid and 0.04 M copper sulfate, pH 5.4. Then these tubes were vortexed for 15-20 seconds to suspend sporangia and then counted with a haemocytometer. These counted spores/sporangia of the pathogen were incubated at 4°C for 1h to push germination. Consequently, these sporangial suspensions and plug of mycelium from pure colony were applied on detached leaves and later, these leaflets were assessed (Frobes et al., 2012; Odilbekov et al., 2014). The isolates were considered more virulent on detached leaves when mean sporulating lesion was ≥1 cm in length when seen with naked eye (Mukalazi et al., 2001; Flier et al., 2007).

 

 

Morphological characterization

Mycelial growth: Agar plug (5 mm) taken from the pure culture and placed in the center of each petri plate had 20 ml of media including Rye Agar A, Corn Meal Agar and Carrot Meal Agar and three replicates for each medium and placed at 18-20°C in the incubator. The pathogen mycelial growth was assessed on 7th day of colony growth with the help of ruler. Then compared with tested media while three mycelial plugs (one from adjacent to original inoculum, 2nd plug among the original inoculum and colony margin, and 3rd plug of margin of colony) cut after 10 days of incubation from unlike positions of the inoculated media plates and gently shacked in 2 ml distilled water to get sporangia suspension. A heamacyto-meter was used to count the numbers of sporangia from three plugs (three petri dishes average used for each medium).

Microscopic observation of sporangia: The morphological characteristics evaluated by using seven-day-old cultures of P. infestans. The sporangia were dislodged in 1 ml of double distilled water added to each plate. The resulting suspension was recovered into a 1.5 ml microcentrifuge tube and dislodged sporangia fixed using 50 µl of acid fuchsin (10 mg acid fuchsin, 100 ml distilled water, 100 ml lactic acid, and the total volume brought to 1.0 ml with double

 

distilled water according to the procedure described by Granke and Hausbeck, 2011. The sporangia of the pathogen were counted through hemocytometer. The sporangia measurements (length, breadth, and pedicel length) were made visually at 200X magnification using a personalized macro. (Torres-Londono, 2016).

Statistical analysis

The morphological characteristics of the isolate compared for each isolate using least significant difference test (LSD) individually. The experimental data subjected to ANOVA using SPSS software v. 19.

Results and Discussion

Sample collection

Late blight identified after visual observing the disease symptoms under field conditions. The earliest symptoms were observed on lower leaves consist of small dark green spots that change from brown-black lesions typically “V” shaped (Hannukkala et al., 2007; Fry, 2008). The samples collection was done from the infected potato fields having sporulation during survey (Figure 3). The phenotypic characteristics of isolates were fluffy cottony mycelia having slight striated patterns; and slow growth rate on different culture media (Figure 4). The culture of P. infestans on Rye agar A at 18oC in dark condition showed white-concentric ring to the cottony colony. A mycelium was hyaline, branched, and coenocyte.

Pathogenicity Assay.

The lesions on detached inoculated leaves were observed after inoculation. The lesion size and amount of leaf damage percentage varied significantly with the isolates. All the inoculated isolates caused disease with same symptoms (described in 3.1) on potato detached leaves. These new infected diseased leaves were again purified for further study.

At the end of the period of inspection, the isolates caused less than 1 cm lesion on detached leaf assay were marked while the best picture concerning the differences between the behaviors of isolates shown in Figure 5. The tendency was the same as the isolate those caused more lesion length caused highest infected percentage area. Therefore, it can be concluded from Pathogenicity assay that the higher the infected area caused by P. infestans isolates on detached leaves the more severe was the infection.

The level of pathogenicity diversity might be arisen as result of selection stress enforced by different cultivated potato cultivars (Blandón-Díaz et al., 2012). High pathotype diversity in northern China has also been reported (Guo et al., 2009).

Mycelial Growth

The mycelia of P. infestans characterized by lack of cross walls and have both asexual and sexual reproduction. The sporangiophores and sporangia of the pathogen appear at asexual reproduction stage. The pathogen’s sporangia are the lemon shaped and developed at the end of these sporangiophores (Figure 6). All collected isolates fit the morphological description with deciduous and semi-papillate sporangia as reported in literature for P. infestans (Erwin et al., 1996).

Sporangial morphology

All isolates produced papillate and deciduous sporangia and the average length of sporangia among the

 

 

isolates collected during 2017 from potato growing areas of Punjab ranged from 14.94 to 47.89 µm, and average breadth of sporangia ranged from 10.44 0 to 23.67 µm. The length/breadth ratio of sporangia ranged from 1.16 to 2.16 amongst the isolates as described by Islam et al. (2005). The isolates during 2017 showed highest length and breadth among six districts were Khb-9 (45.44 x 21.22 μm), Srg-1 (42.89 x 19.89 μm), Shl-10 (47.89 x 23.22 μm), Oka-13 (47.83 x 23.56 μm), Jhg-10 (47.56 x 23.57 μm) and Cht-6 (45.28 x 21.61 μm) (Figure 7). During 2018 tested isolates against morphological characteristics, among all districts, isolate Jhg-22 showed highest length (46.64 μm) and breadth (22.17 μm) followed by Shl-25 (46.59 μm) and breadth (22.61 μm), Oka-24 (46.31 x 21.72 μm) while lowest value measured by Cht-14 (16.05 x 13.39 μm). The isolate mean values compared, sorted within the district and presented in graphs (Figure 8).

The length and breadth (46.25 x 22.61 μm) was the highest value recorded in case of Khb-21 isolate from Khushab district while Srg-14, Shl-25, Oka-24, Jhg-22, Cht-18 (4.83 x 22.17 μm, 46.59 x 22.61 μm, 46.31 x 21.72 μm, 46.64 x 22.17 μm and 44.06 x 23.05 μm) from Sargodha, Sahiwal, Okara, Jhang and Chiniot respectively (Figure 8). It observed during both the years that isolates caused the highest values of length and breadth selected isolates from previous aggressiveness experiment by Raza et al. (2019). They observed that more aggressive isolates had the highest values of morphological characteristics as compared to other and the same trend observed in the current study.

Culture Media Evaluation

Variation in the colony color, the margin of the colony on three different solid media adds the vital information, which may help in taxonomic identification of P. infestans. It showed a difference in growth ability and sporulation ability on the selected media. In the present study, it was found that Rye agar media showed the best sporulation of the fungus, followed by Cornmeal media, and the least growth was recorded in case of carrot agar media.

The overall data of six districts revealed that during 2017 study, Rye agar medium had significantly the highest growth of Oka-13 (70.33 mm) followed by Jhang-10 (69.67 mm), Shl-10 (69.33 mm) while the least growth was observed by Oka-7 having 34.67 mm. The spores were produced more on Rye agar media by isolate Oka-13 (228.67 per square cm) as compared to other media used (Figure 9).

Generally, the isolates showed a similar pattern of growth on the second year 2018 and here presented an overview of top three isolates of each district that Rye agar media was the best media for the radial growth e.g Khushab district isolates were Khb-21 (65 mm) followed by Khb-11 (57.67 mm) and Khb-15 (49.33 mm). Among Sargodha district isolates, Srg-14 (65 mm), Srg-24 (57.67 mm), Srg-21 (51 mm). In the case of Sahiwal district isolates, Shl-25 (67 mm),

 

 

Shl-20 (62.33 mm), Shl-24 (56 mm). The greatest growth was recorded from Okara isolates was Oka-24 (68 mm), Oka-21 (60 mm) and Oka-19 (50 mm). Radial growth of Isolates collected from Jhang district ranged from 36.33 mm - 44.33 mm against Jhg-19 and Jhg-22 respectively on tested media at 18°C. Similar trends were also noted in district in isolate Cht-18 from Chiniot showed good colony growth (66.67 mm) followed by Cht-23 (57 mm) and Cht-16 (54.67 mm) (Figure 10). Indeed, all isolates among 6 districts studied showed almost similar response towards Rye agar media while somewhat less on media containing Corn meal agar and carrot agar media. The smallest radial growth obtained on Carrot agar medium may due to incompatibility with pathogen requirements.

 

 

Potato late blight is the most economical and significant plant disease worldwide. Morphological identification of this plant pathogen is an important subject that caused high destruction on potato. There are several factors that possible affecting the purified of the isolations such as the presence of contaminants and the environmental conditions. The Phytophthora genus can be isolated from a small proportion of the soil samples taken by the infected leaves (Vettraino et al., 2001).

However, isolation of P. infestans from the infected sample requires substantial time with using the selective media (Yamak et al., 2002). A major concern to get a pure culture of Phytophthora is possible the presence of fast-growing organisms such as Pythium and other genera of fungi, which affected the growth of Phytophthora (Canaday and Schmitthenner, 1982). As well, the material of potato seed can affect the fungal co mmunity in the fields (Runno-Paurson et al., 2010b, Drenth et al., 2001; Lal et al., 2015). In the present study, morphological characters of sporangial size was undistinguished which was found to be high values of aggressive isolates. Sopee et al. (2011a) revealed a variation on size of the pedicel, sporangium and oospore of P. infestans isolates and analyzed on the relationship of the size of these propagules with metalaxyl sensitivity.

Interestingly, the difference in the culture nutrient for the plant fungal pathogens is showing some variability among races and isolates of the same pathogen (Lehtinen et al., 2007). Different techniques are often used for the revealing of Phytophthora spp. within the fungal population (Gallegly and Hong, 2008). The primary obstacle of contamination led to the use of selective medium to enhance the growth of the desired pathogens (Kumbar, 2017; Fry, 2008; Ristaino, 2012; Grünwald et al., 2011; Tian et al., 2016; Ho, 2018). In the current study, variation in the growth pattern of the races of Phytophthora in different culture media was obtained. The variations of the isolates were based on morphological characteristics, which were detected in this study. The characteristic of morphological may be relevant with changing in the temperatures that possible degradation the components of culture media (Cooke and Lees, 2004).

Several growth media such as Rye Agar media, Corn meal agar and Carrot Agar media are very important for the growth of the P. infestans (Peters et al., 1998). Rye Agar media used to prepare P. infestans culture media (Peters et al., 1998). Other cultural media used to grow and sporulate of P. infestans but inappropriate for many isolates (Savage et al., 1968). It is using a specific media for getting a typical growth of P. infestans that can help to study this organism and capability to cause high damage for the potato fields.

On the other hand, carrot agar media does not support abundant sporangial production as compared to other media (Erwin and Ribeiro, 1996). Lower growth of the fungus in carrot meal and cornmeal agar medium may return into three reasons, first, not contain the substances supporting the growth of pathogens, second, the substances may be destroyed during autoclaving and when they integrated into the medium, and third, the substances complexed. Two cultural media such as carrot meal and cornmeal agar may be lacking the suitable nutrient content or not ready (Complex nutrient) for growing and sporulation of P. infestans that caused poor growth. This species as P. infestans may be lacking some enzymes that are degrading the nutrient from complex to be more simple nutrients. For the highest growth and appearance, nutrient contents are very significant. Based on observations by different researchers, carbohydrates are much necessary for fungal growth and its sporulation (Kumbar, 2017). This present investigation revealed that Rye Agar medium is considered as the best medium to grow of P. infestans and the next medium is a cornmeal agar. While carrot agar medium supports the sporulation of P. infestans causes of potato late blight. This detected a suitable medium to grow and reproduction of P. infestans that will utilize in studying of different sides of this species including the molecular study, and management strategies of the blight late disease.

Novelty Statement

Phytophthora infestans is a destructive plant pathogen in the tropical and the sub-tropical areas of worldwide. Phenotypic identification of pathogens is very helpful to find successful disease control strategies. Knowledge of variability in morphological, cultural parameters of P. infestans is still scarce. Therefore, the determination of morphological characteristics may add knowledge of the taxonomic behavior of pathogen which ultimately leads towards its control.

Author’s Contribution

Waqas Raza: Executed the field visits, wrote and finalized the manuscript.

Muhammad Usman Ghazanfar: Conceived the idea and facilitated guided and supervised the experiment.

Muhammad Asif: Guided for experiments.

Ikram Ul Haq: Help in data analysis.

Muhammad Zakria: Co-supervised the experiments.

Laith Khalil Tawfeeq Al-Ani: Guided and help for data analysis.

Conflict of Interest

The authors declare that there is no conflict of interest to publish the article.

References

Adl, S.M., A.G.B. Simpson, M.A. Farmer, A.A. Andersen, R.O. Anderson, J.R. Barta, S.S. Bowser, G. Brugerolle, R.A. Fensome, S. Fredericq, T.Y. James and S. Karpov. 2005. The New Higher Level Classification of Eukaryotes with Emphasis on the Taxonomy of Protists. Journal of Eukaryotic Microbiology. 52: 399-451. https://doi.org/10.1111/j.1550-7408.2005.00053.x

Arora, R.K., S. Sanjeev, B.P. Singh. 2014. Late blight disease of potato and its Management. Potato Journal. 4: 16-40.

Birch, P.R.J., G. Bryan, B. Fenton, E.M. Gilroy, I. Hein, J.T. Jones, A. Prashar, M.A. Taylor, L. Torrance and I.K. Toth. 2012. Crops that feed the world 8: Potato: are the trends of increased global production sustainable? Food Security. 4: 477–508. https://doi.org/10.1007/s12571-012-0220-1

Blandón-Díaz, J.U., A.K. Widmark, A. Hannukkala, B. Andersson, N. Högberg and J.E. Yuen. 2012. Phenotypic variation within a clonal lineage of Phytophthora infestans infecting both tomato and potato in Nicaragua. Phytopathology. 102(3); 323-330. https://doi.org/10.1094/PHYTO-02-11-0033

Bower, J.H., F.N. Martin, P.W. Tooley and E.D.M.N. Luz. 2007. Genetic and morphological diversity of temperate and tropical isolates of Phytophthora capsici. Phytopathology. 97: 492-503. https://doi.org/10.1094/PHYTO-97-4-0492

Canaday, C. and A. Schmitthenner. 1982. Isolating Phytophthora megasperma f. sp. glycinea from soil with a baiting method that minimizes Pythium contamination. Soil Biology and Biochemistry. 14: 67–68. https://doi.org/10.1016/0038-0717(82)90079-7

Caspary, R. 1852. Peronospora infestans (Mont.) Caspary. Klotzsch Herbar. Mycol no. 1879.

Caten, C.E. and J.L. Jinks. 1968. Spontaneous variability of single isolates of Phytophthora infestans. I. Cultural variation. Canadian Journal of Botany. 46: 329-348. https://doi.org/10.1139/b68-055

Cooke, D.E.L. and A.K. Lees. 2004. Markers, old and new, for examining Phytophthora infestans diversity. Plant Pathology. 53: 692-704. https://doi.org/10.1111/j.1365-3059.2004.01104.x

Cooke, L.R., H.T.A.M. Schepers, A. Hermansen, R.A. Bain, N.J. Bradshaw, F. Ritchie, D.S. Shaw and A. Evenhuis. 2011. Epidemiology and integrated control of potato late blight in Europe. Potato Research. 54: 183-222. https://doi.org/10.1007/s11540-011-9187-0

De Bary, A. 1876. Researches into the nature of the potato–fungus, Phytophthora infestans. Journal of the Royal Agricultural Society. 12: 239–269.

Drenth, A. and B. Sendall. 2001. Practical guide to detection and identification of Phytophthora. CRC for Tropical Plant Protection, Brisbane, Australia 1-41.

Erselius, L.J. and D.S. Shaw. 1982. Protein and enzyme difference between Phytophthora palmivora and P. megakarya: evidence for self-fertilization in pairings of the two species. Transactions of the British Mycological Society 78: 227-238. https://doi.org/10.1016/S0007-1536(82)80005-3

Erwin, D.C. and O.K. Ribeiro. 1996. Phytophthora Diseases Worldwide. American Phytopathological Society Press, St. Paul, MN.

Flier, W.G., L.P.N.M. Kroon, A. Hermansen, H.M.G. Van Raaij, B. Speiser, L. Ta mm and S. Wilcockson. 2007. Genetic structure and pathogenicity of populations of Phytophthora infestans from organic potato crops in France, Norway, Switzerland and the United Kingdom. Plant Pathology. 56(4): 562-572. https://doi.org/10.1111/j.1365-3059.2007.01571.x

Flier, W.G., L.J. Turkensteen, G.B.M. Van Den Bosch, P.F.G. Vereijken, A. Mulder. 2001. Differential interaction of Phytophthora infestans on tubers of potato cultivars with different levels of blight resistance. Plant Pathology. 50: 292-301. https://doi.org/10.1046/j.1365-3059.2001.00574.x

Forbes, G.A. 2012. Using host resistance to manage potato late blight with particular reference to developing countries. Potato Research. 55(4): 205-216. https://doi.org/10.1007/s11540-012-9222-9

Fry, W.E. 2008. Phytophthora infestans: The plant (and R gene) destroyer. Molecular Plant Pathology. 9: 385-402. https://doi.org/10.1111/j.1364-3703.2007.00465.x

Gallegly, M.E. and C. Hong. 2008. Phytophthora: identifying species by morphology and DNA fingerprints. American Phytopathological Society (APS press), St. Paul, Minnesota, USA.

Goth, R.W. 1981. An efficient technique for prolonged storage of Phytophthora infestans. American Journal of Potato Research. 58: 257-260. https://doi.org/10.1007/BF02853907

Gotoh, K., S. Akino, M. Kato, N. Kondo and S. Naito. 2005. Characterization of some Asian isolates of Phytophthora infestans. Plant Pathology. 54: 733–739. https://doi.org/10.1111/j.1365-3059.2005.01286.x

Granke, L.L. and M.K. Hausbeck. 2011. Variation in phenotypic characteristics of Phytophthora capsici isolates from a worldwide collection. Plant Disease. 95: 1080–1088. https://doi.org/10.1094/PDIS-03-11-0190

Granke, L.L., S.T. Windstam, H.C. Hoch, C.D. Smart and M.K. Hausbeck. 2009. Dispersal and movement mechanisms of Phytophthora capsici sporangia. Phytopathology. 99: 1258–1264. https://doi.org/10.1094/PHYTO-99-11-1258

Grünwald, N.J., F.N. Martin and M.M. Larsen. 2011. Phytophthora–ID.org: A sequence–based Phytophthora identification tool. Plant Disease. 95: 337–342. https://doi.org/10.1094/PDIS-08-10-0609

Guo, J., T. Van Der Lee, D.Y. Qu, Y.Q. Yao, X.F. Gong, D.L. Liang and F. Govers. 2009. Phytophthora infestans isolates from Northern China show high virulence diversity but low genotypic diversity. Plant Biology. 11(1): 57-67. https://doi.org/10.1111/j.1438-8677.2008.00159.x

Hannukkala, A.O., T. Kaukoranta, A. Lehtinen, A. Rahkonen. 2007. Late-blight epidemics on potato in Finland, 1933–2002; increased and earlier occurrence of epidemics associated with climate change and lack of rotation. Plant Pathology. 56: 167-176. https://doi.org/10.1111/j.1365-3059.2006.01451.x

Hartman, G.L. and Y.H. Huang. 1995. Characteristics of Phytophthora infestans isolates and development of late blight on tomato in Taiwan. Plant Disease. 79: 849-852. https://doi.org/10.1094/PD-79-0849

Haverkort, A.J. 2008. Societal costs of late blight in potato and prospects of durable resistance through cisgenic modification. Potato Research. 51: 47–57. https://doi.org/10.1007/s11540-008-9089-y

Ho, H.H. 2018. The taxonomy and biology of Phytophthora and Pythium. Journal of Bacteriology and Mycology. 6: 00174. https://doi.org/10.15406/jbmoa.2018.06.00174

Hong, C., P.A. Richardson, P. Kong, P. 2008b. Pathogenicity to ornamental plants of some existing species and new taxa of Phytophthora from irrigation water. Plant Disease. 92: 1201–1207. https://doi.org/10.1094/PDIS-92-8-1201

Islam, S.Z., M. Babadoost, K.N. Lambert, A. Ndeme and H.M. Fouly. 2005. Characterization of Phytophthora capsici isolates from processing pumpkin in Illinois. Plant Disease. 89: 191-197. https://doi.org/10.1094/PD-89-0191

Jaimasit, P. and W. Prakob. 2011. Characterization of Phytophthora infestans population in potato crops from Chiang mai and Tak provinces. Journal of Agricultural Technology. 7: 431- 439.

Kandel, K.P. 2014. Transcriptomic studies of the early stages of potato infection by Phytophthora infestans (Doctoral dissertation, University of Dundee).

Kiptoo, J., A. Abbas, A.M. Bhatti, H.M. Usman, M.A. Shad, M. Umer, M.N. Atiq, S. M. Alam, M. Ateeq, M. Khan, N. W. Peris, Z. Razaq, N. Anwar and S. Iqbal. 2021. Rhizoctonia solani of potato and its management: a review. Plant Protection. 5(3): 157-169. https://doi.org/10.33804/pp.005.03.3925

Kumbar, B. 2017. Standardization of specific media for Phytophthora Infestans. Global Journal of Bio-Science and Biotechnology. 6: 374-376.

Lal, M., C. Lal, S. Yadav, S.B. Gunjan, S.K. Kaushik, S. Sharma. 2015. Biological characterization, mt haplotyping, and chemical management of Phytophthora infestans causing late blight of potato. Int. J. Agricult. Stat. Sci. 11(1): 259-266.

Lehtinen, A., A. Hannukkala, B. Andersson, A. Hermansen, V.H. Le, R. Naerstad and J. Yuen. 2008. Phenotypic variation in Nordic populations of Phytophthora infestans in 2003. Plant Pathology. 57: 227-234. https://doi.org/10.1111/j.1365-3059.2007.01739.x

Lehtinen, A., A. Hannukkala, T. Rantanen and L. Jauhiainen. 2007. Phenotypic and genetic variation in Finnish potato-late blight populations, 1997-2000. Plant Pathology. 56: 480-491. https://doi.org/10.1111/j.1365-3059.2006.01556.x

Mahr, N.A. 2021. Comparative efficacy of ten co mmercial fungicides for the control of Rhizoctonia solani, the cause of black scurf disease of potato. Plant Protection. 5(3): 149-156. https://doi.org/10.33804/pp.005.03.3930

Martin, F.N., Z.G. Abad, Y. Balci, K. Ivors. 2012. Identification and detection of Phytophthora: Reviewing our progress, identifying our needs. Plant Disease. 96: 1080–1103. https://doi.org/10.1094/PDIS-12-11-1036-FE

Medina, M.V. 1999. Comparison of different culture media on the mycelial growth, sporangia and oospore production of Phytophthora infestans. American Journal of Potato Research. 76: 121-125. https://doi.org/10.1007/BF02853576

Montagne, C. 1845. Observations sur la maladie des pomenes de terre. Bull. De la Soc. Philoatique de Paris. 13: 312–314.

Mrázkova, M.K. Černý, M. Tomšovs ký and V. Strnadová. 2011. Phytophthora plurivora Jung T, Burgess TI and other. Phytophthora species causing important diseases of ericaceous plants in the Czech Republic. Plant Protection Science. 47: 13–19. https://doi.org/10.17221/3108-PPS

Mukalazi, J., E. Adipala, T. Sengooba, J.J. Hakiza, M. Olanya and H.M. Kidanemariam. 2001. Metalaxyl resistance, mating type and pathogenicity of Phytophthora infestans in Uganda. Crop Protection. 20(5): 379-388. https://doi.org/10.1016/S0261-2194(00)00145-9

Nisa, T., M.I. Haq, T. Mukhtar, M.A. Khan and G. Irshad. 2022. Incidence and severity of co mmon scab of potato caused by Streptomyces scabies in Punjab, Pakistan. Pakistan Journal of Botany. 54(2): 723-729. https://doi.org/10.30848/PJB2022-2(36)

Odilbekov, F., U. Carlson-Nilsson and E. Liljeroth. 2014. Phenotyping early blight resistance in potato cultivars and breeding clones. Euphytica. 197(1): 87-97. https://doi.org/10.1007/s10681-013-1054-4

Peters, R.D., H.W. Platt and R. Hall. 1998. Long-term survival of Phytophthora infestans in liquid media prepared from autoclaved seeds. Canadian Journal of Plant Pathology. 20: 165-170. https://doi.org/10.1080/07060669809500422

Pliakhnevich, M. And V. Ivaniuk. 2008. Aggressiveness and metalaxyl sensitivity of Phytophthora infestans strains in Belarus. Zemdirbyste. 95(3): 379-387.

Raza, W. and M.U. Ghazanfar. 2019. General Research Methods to Deal with Phytophthora infestans in Laboratory Conditions. EC Agriculture. 5: 2008-2012.

Reis, A., C.D. Smart, W.E. Fry, L.A. Maffia and E.S.G. Mizubuti. 2003. Characterization of isolates of Phytophthora infestans from southern and southeastern Brazil from 1998 to 2000. Plant Disease. 87: 896-900. https://doi.org/10.1094/PDIS.2003.87.8.896

Ribeiro, O.K. 2013. A historical perspective of Phytophthora. Phytophthora: a global perspective 2, 1. https://doi.org/10.1079/9781780640938.0001

Ristaino, J.B. 2012. A lucid key to the co mmon Phytophthora species. USA: APS Press. 28.

Runno-Paurson, E., T. Re mmel, A. Ojarand, A. Aav and M. Mänd 2010b. The structure of the Phytophthora infestans population in organic and conventional crops in Estonia. European Journal of Plant Pathology. 128: 373-383. https://doi.org/10.1007/s10658-010-9659-0

Sanyong, S., C. Chamswang, P. Ha mmerlink and A. Sukthumrong. 1993. Identification of race and mating type of Phytophthora infestans, a causal agent of potato late blight. Proceedings of 31st Kasetsart University Annual Conference in Agriculture Section. Kasetsart University, 3-6 February, Bangkok. 542-550. (In Thai).

Savage, E.J., C.W. Clayton, J.H. Hunter, J.A. Brenneman, C. Laviola and M.E. Gallegly. 1968. Homothallism, heterothallism, and interspecific hybridization in the genus Phytophthora. Phytopathology. 58: 1004-1021.

Schumann, G.L. and C.J. D’Arcy. 2000. Late blight of potato and tomato. The Plant Health Instructor. https://doi.org/10.1094/PHI-I-2000-0724-01

Schumann, G.L. and C.J. D’Arcy. 2007. Essential Plant Pathology. APS Press, St. Paul, Minnesota.

Sopee, J. 2008. Modification of culture medium for growth and sporulation of Phytophthora infestans (Mont.) de Bary. Phytopathology. 98: S148.

Sopee, J., T. Mekmok, C. Kanthawong, S. Pinitkul, U. Saensabai and P. Chiampiriyakul. 2011a. Micro-morphology and genetic variation of Phytophthora infestans isolated from potato. The proceeding of 49th Kasetsart University Annual Conference (Subject: Plant). Kasetsart University, Bangkok. (In Thai, with English abstract).

Sujkowski, L.S., G. Parra, M.L. Gumpertz and J.B. Ristaino. 1999. Temporal dynamics of Phytophthora blight on bell pepper in relation to the mechanisms of dispersal of primary inoculum of Phytophthora capsici in soil. Phytopathology. 90: 148–156. https://doi.org/10.1094/PHYTO.2000.90.2.148

Tian, Y.E., J.L. Yin, J.P. Sun, Y.F. Ma, Q.H. Wang, J.L. Quan and W.X. Shan. 2016. Population genetic analysis of Phytophthora infestans in northwestern China. Plant Pathology. 65: 17-25. https://doi.org/10.1111/ppa.12392

Torres-Londono, G.A. 2016. Morphological Characterization, Virulence, and Fungicide Sensitivity Evaluation of Phytophthora Palmivora. PhD thesis, Michigan State University. p.129.

Tyler, B.M. 2002. Molecular basis of recognition between Phytophthora pathogens and their hosts. Annual Review of Phytopathology. 40: 137–67.

Vettraino, A.M., G. Natili, N. Anselmi and A. Vannini. 2001. Recovery and pathogenicity of Phytophthora species associated with a resurgence of ink disease on Castanea sativa in Italy. Plant Pathology. 50: 90–96. https://doi.org/10.1046/j.1365-3059.2001.00528.x

Wang, B., Y. Ma, Z. Zhang, Z. Wu, Y. Wu, Q. Wang and M. Li. 2011. Potato viruses in China. Crop Protection. 30: 1117–1123. https://doi.org/10.1016/j.cropro.2011.04.001

Yamak, F., T.L. Peever, G.G. Grove, R.J. Boal. 2002. Occurrence and identification of Phytophthora spp. pathogenic to pear fruit in irrigation water in the Wenatchee River Valley of Washington state. Phytopathology. 92: 1210–1217. https://doi.org/10.1094/PHYTO.2002.92.11.1210

Young, G.K. 2007. Competitive selection within Phytophthora infestans populations from Northern Ireland and Michigan. Ph. D. Thesis, The Queen’s University of Belfast, 300 pp.

Zhang, S., N. Yang and H. Huang. 2017. Study on grain demand structure change in Jilin province and its contribution rate to China’s food security. Advances in Computer Science Research. 59: 247–251.