Direct Isolation Method for Tomato-Infecting Phytophthora infestans Metode Isolasi Langsung untuk Phytophthora infestans yang Menginfeksi Tanaman Tomat

Infection of Phytophthora infestans at high altitudes in tropical regions causes symptoms of tomato late blight throughout the year. Even though it is very easy to find in the field, P. infestans is often very difficult to isolate aseptically in the laboratory. This study aims to evaluate direct isolation techniques that can increase the success of isolating P. infestans . Isolation was carried out on a non-specific medium, consisting of potato dextrose agar (PDA), corn meal agar (CMA), oatmeal agar (OMA), and water agar (WA) with three alternative types of antibiotics, i.e. chloramphenicol, amoxicillin, and rifampicin. Observations were carried out to determine the effect of the medium on sporangia induction and the age of the original colony’s growth in CMA and OMA media. The results showed that the isolation of P. infestans using the direct method was successfully carried out on non-specific PDA, CMA, OMA and WA medium. The highest isolation success rate was obtained on CMA medium with the addition of 50 mg L -1 rifampicin. The fastest sporangia induction (8 days) was shown by colonies grown on OMA medium with the addition of 50 mg L -1 rifampicin based on the category of abundant sporangia after 20 days of incubation. Rejuvenation of P. infestans colonies for research purposes in the laboratory is recommended to be carried out routinely twice a month. This research provides practical guidance for understanding the bioecology of P. infestans infecting tomato plants, especially for further study on oomycetes fungi.


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Phytophthora infestans (Mont.)de Bary, the most invasive pathogen causing late blight disease, has become a constraint to many horticultural crops, including potatoes and tomatoes (Nelson 2008;Bush et al. 2012;Nowicki et al. 2012;Dangi et al. 2020).The symptoms of P. infestans infection are easily found in tropical countries like Indonesia, particularly in tomato-producing regions in high land with cold temperatures.The typical symptoms manifest on the leaves and fruits, resulting in increased losses in marketable production.Nelson (2008) explains that this pathogen can spread rapidly in cool and wet conditions, causes plant death within a few days, and results in total yield loss.In contrast to studies on potatoes, there have been relatively few reports of this disease on tomatoes in Indonesia over the past five years.
The current most common method to isolate and culture P. infestans is based on zoospores collection.In these methods, zoospores were collected from infected leaf tissues or rotten fruits from the field (Griffith et al. 1995;Oyarzun et al. 1998;Tumwine et al. 2000).However, these methods have several drawbacks that lead to a poor isolation success rate.Since it takes time to travel from the field to the laboratory, it is possible that sporangia on the leaf surface were discharged into the atmosphere and mixed with other pathogens.Moreover, according to Plancarte et al. (2017), Phytophthora spp.should not be isolated from necrotic tissue due to the highly potential presence of saprophytic bacteria and fungi.In an attempt to isolate P. infestans in the field, Suzuki et al. (2019) applied a method in which the infected leaf surface was taped onto the agar medium.Despite the use of a specific agar medium (Rye-B agar), the isolation failure rate remained high due to contamination by diverse microorganisms.
It is quite challenging to culture P. infestans axenically due to its near-obligate biotrophic nature, making it prone to agar culture contamination (Griffith et al. 1995).This pathogen and bacteria coexist in plant-microbe interactions.However, the mechanism by which P. infestans interacts with the microbes in its environment to develop ecological adaptation remains unexplained (Berendsen et al. 2012;Wang et al. 2020).Kong and Hong (2016) have demonstrated that bacteria are essential sources of virulence signals that help Phytophthora sp.infect plants.We therefore hypothesize that one of the main challenges facing scientists studying this oomycete is its isolation process.Another area for improvement with culture is obtaining the expensive and hard-to-find components for the specific medium (e.g.V8 agar or rye agar).To isolate P. infestans from the field, we evaluated some non-specific medium in addition to a simple isolation method.It is essential to add antibiotics to the agar medium during the isolation process to suppress the growth of various microorganisms.Oomycetes researchers have extensively studied the use of different antibiotics on agar medium in their quest for optimal isolation methods.A notable study by Masago et al. (1976) demonstrated the successful suppression of bacteria, non-pythiaceous fungi, and twelve species of Pythium spp.when isolating terhadap pertumbuhannya pada medium CMA dan OMA.Hasil pengujian menunjukkan bahwa isolasi P. infestans dengan metode langsung berhasil dilakukan pada medium non-spesifik PDA, CMA, OMA dan WA dengan tingkat keberhasilan isolasi tertinggi diperoleh pada medium CMA dengan penambahan 50 mg L -1 rifampicin.Induksi sporangia tercepat (8 hari) ditunjukkan oleh koloni yang ditumbuhkan pada medium OMA dengan penambahan 50 mg L -1 rifampicin, yaitu berdasarkan pada kategori jumlah sporangia yang berlimpah setelah 20 hari inkubasi.Peremajaan koloni P. infestans untuk keperluan penelitian di laboratorium disarankan untuk dilakukan secara rutin dua kali dalam sebulan.Penelitian ini memberikan panduan praktis untuk memahami bioekologi P. infestans yang menginfeksi tanaman tomat, terutama bagi para peneliti oomycetes dalam mengisolasi dan mengkaji patogen ini lebih lanjut.
Kata kunci: busuk daun, inkubasi, medium non-spesifik, rifampisin, sporangia Phytophthora spp.from soil and infected plant tissues.Their findings clearly show the effectiveness of nystatin, rifampicin, and ampicillin in inhibiting various Phytophthora spp.without affecting mycelial growth or spore germination.
This research was conducted at the Plant Mycology Laboratory, Department of Plant Protection, IPB University.We sampled and isolated P. infestans directly in the tomato fields from IPB Experimental Station at Pasir Sarongge, Pacet District, Cianjur Regency, West Java.This field location was ideal for direct isolation because it had a high prevalence of P. infestans infection for the last three years.
The field visit was conducted in the early morning, right after rainfall the previous day.This made it easier to identify symptoms in the form of relatively new lesions on the leaves of infected tomato plants (Figure 1a and 1b).Direct sporangia sampling was carried out on various medium including potato dextrose agar (PDA, HiMedia Laboratories Pvt.Ltd), corn meal agar (CMA, HiMedia Laboratories Pvt.Ltd), oat meal agar (OMA: rolled oats and Bacto agar), and water agar (Bacto agar, Difco Laboratories, USA) which were supplemented with three types of antibiotics, i.e. chloramphenicol (500 mg L -1 ), rifampicin (50 mg L -1 ), and amoxicillin (100 mg L -1 ).Antibiotics and medium were used to stimulate the growth of sporangia.In each agar plate, sporangia were obtained by scraping the sterile inoculation loop from the freshly created lesion and sealed (Figure 1c).The samples were transported to the laboratory and incubated for 48 hours at 16 ℃.The isolated fungal colonies were identified by morphological observation, as described in Drenth and Sendall (2001).Additionally, the growth of the colonies was assessed every 12 to 72 hours.
Compared to the other three agar medium, CMA supplemented with 50 mg L -1 rifampicin yielded the best results (Table 1).However, on the same media, the fungi failed to grow with the addition of 500 mg L -1 chloramphenicol and only 1 plate of the fungi grew with the addition of 100 mg L -1 amoxicillin.Isolation using OMA also provides quite effective results when 50 mg L -1 rifampicin or 100 mg L -1 amoxicillin is added.Meanwhile, the use of PDA and water agar as a medium only gives a low rate of success.
Figure 2 shows the pure colonies of P. infestans on PDA, CMA, and OMA supplemented with 50 mg L -1 rifampicin on each medium.These results prove that rifampicin is the most effective antibiotic for isolating P. infestans.Rifampicin inhibits bacterial DNA-dependent RNA synthesis and prevents the production of host bacterial proteins, targeting mycobacteria and gramnegative bacteria (Sarker et al. 2020).In our study, the isolates were successfully sub cultured on PDA, CMA, and OMA.Aseptate hyphae were observed, but no sporangia were visible.To induce P. infestans sporangia production on agar medium and the rate of its induction, CMA or OMA combined with rifampicin or amoxicillin was examined as the recommended options during the isolation stage.The optimal medium for inducing sporangia production at 16 ℃ in the dark was OMA supplemented with 50 mg L -1 rifampicin.The incubation period was the shortest (eight days) (Table 2).Furthermore, after 20 days of incubation, a high density of sporangia was formed on OMA supplemented with 50 mg L -1 rifampicin (Figure 3b).In contrast, sporangia density was observed to be scarce on CMA supplemented with 50 mg L -1 rifampicin (Figure 3a).
Table 1 Comparison of four agar medium and three antibiotics for direct isolation of Phytophthora infestans from infected leaves in the field Our further examination involved subculturing the various ages of the original P. infestans colonies (8, 13, 18, 23, and 28-dayold colonies) to ascertain how frequently these oomycetes should be sub-cultured to a fresh medium and which age of the original colony is still ideal for sub-culture.We sub-cultured each colony of different ages onto a new agar medium, with ten replicates on CMA and OMA.We observed the colonies' diameter while incubating for up to 72 hours at room temperature (22 ℃).P. infestans colonies in both CMA and OMA expanded quickly upon subculturing them from 8 and 13 days of original colonies (Figure 4 and Figure 5).New colonies that are 18, 23, or 28 days old either exhibit abnormal development or fail to revive.Subculturing P. infestans isolates from colonies that are 23 or 28 days old (both CMA and OMA) carries an increased risk of bacterial contamination during the incubation period.This observation demands that biweekly subculturing steps be carried out if the cultures are to be utilized for routine tests before being stored for an extended period.
According to this study, direct isolation with CMA supplemented with 50 mg L -1 rifampicin was strongly recommended.Sporangia sampling should be conducted in the early morning since it provides maximum relative humidity and it is easier to discover fresh lesion symptoms.Furthermore, colonies that successfully expanded in the first one to two days should be sub-cultured immediately to the fresh medium to avoid bacterial contamination.This approach saves time and helps the oomycetes researcher face challenges with the shortcomings of a specific growing medium.
In conclusion, this study has successfully presented new insights that have practical implications for understanding the bioecology of P. infestans infecting tomato plants.These insights enable researchers to isolate P. infestans more efficiently and handle the isolates in the laboratory for continuous

Figure 1
Figure 1 Direct isolation of Phytophthora infestans from lesion of tomato late blight in the field.a-b, infected leaf with sporangia mass; and c, inoculating the sporangia onto corn meal agar (CMA) medium supplemented with 50 mg L -1 rifampicin after picking them up with sterile inoculation loop.c a b Figure 2 Two-day-old colony of Phytophthora infestans on agar medium supplemented with 50 mg L -1 rifampicin.a, corn meal agar (CMA); b, potato dextrose agar (PDA); and c, oatmeal agar (OMA).c a b , corn meal agar; OMA, oatmeal agar a Data followed by the different letter show significant difference by Tukey test (α 5%).Data are given as mean ± SE; b Based on 10× observations from 5 mm diameter of discs cut from random site of colony plate.

Table 2
Induction of sporangia formation on corn meal agar (CMA) and oatmeal agar (OMA) media incubated at 16 ℃