Comparison of growth, viability, sporulation, and virulence of Phytophthora capsici isolated from black pepper (Piper nigrum L.) on various media.

Black pepper (Piper nigrum L.) is an important agricultural crop in Indonesia. However, black pepper production in Indonesia is limited by plant pathogen infection, especially Phytophthora capsici, which causes Foot Rot in pepper plants. This pathogen contributes to a high decline in black pepper productivity. This study aims to determine the proper media composition for P. capsici growth based on the mycelial growth, viability, sporulation, and virulence of P. capsici on pepper leaves. The media used in this study were potato dextrose agar (PDA), martin agar (MA), carrot agar (CA), water agar (WA), corn meal agar (CMA), soil extract agar (SEA), and vegetable 8 (V8). The results showed that CMA media was the most optimal medium for P. capsici growth, as indicated by the size of the mycelium with a diameter of 8.00 cm on the fifth day after inoculation. The viability of mycelium of P. capsici was best found on PDA, CMA, and V8 media. Meanwhile, the optimum sporulation and virulence of P. capsici occurred on SEA media, characterized by zoospore production of 5.42x106/ml and symptom areas on leaves of 18.61 cm2 and 16.69 cm2.


Introduction
The productivity of pepper as a leading export commodity in Indonesia is often limited by the disease.Pseudofungi Phytophthora capsici Leon is an important pathogen on pepper plants that causes foot rot disease.In contrast to fungi in general, P. capsici as a soil-borne pathogen can grow optimally in watersaturated soil conditions, is sensitive to nutrients and the growing media environment, and is easily carried through water, plant residues, and soil.This fungus can also form oospores or chlamydospores as resting structures that is able to survive for decades in the soil, even in extreme environmental conditions [1].
Foot rot disease can reduce pepper productivity by around 30-40%.Pseudofungi Phytophthora capsici Leon is a vital pathogen causing foot rot disease in chili.P. capsici is a soil-borne pathogen that can survive effectively in water-saturated soil conditions, is sensitive to nutrients and the growing media 1297 (2024) 012027 IOP Publishing doi:10.1088/1755-1315/1297/1/012027 2 environment, and is easily spread by water, plant waste, and soil, in opposite to fungi in general.These fungi can also produce oospores or chlamydospores, which are resting structures that can survive in the soil for decades, even in harsh environments [1] The growing media for P. capsici isolates that are often used in laboratories are potato dextrose agar (PDA), corn meal agar (CMA), and vegetable-8 (V8) media with the addition of the antibiotic ampicillin which functions to inhibit bacterial growth [2] However, the effectiveness of media storage in maintaining colony mycelium growth, pathogenicity, and virulence of P. capsici decreased during storage.This causes P. capsici isolate subcultures to be difficult to purify again on PDA, CMA, and V8 media, causing P. capsici isolates cannot be used in the long term [3].
Various control technologies applied in laboratories, greenhouses, and fields are required to develop a comprehensive strategy.These tests require large quantities of zoospores and storage media with long shelf life.[4] reported that a good storage medium for maintaining the pathogenicity of P. palmivora is sterile water.Therefore, an in-depth study is necessary to determine the media that are capable of sustaining hyphal growth, producing zoospores, and the pathogenicity of P. capsici.

2.1.
Sampling methods The sampling of pepper plants with BSR symptoms was carried out by taking samples of pepper leaves in areas between the healthy and diseased leaves.The pepper leaf samples taken have a special feature, particularly the ragged edges of the leaves.

Preparation of P. capsici growth media
The media used in this study were PDA, CMA, WA, CA, MA, and V8 media referring to the method used by [5] that was modified.Besides the common media, soil extract agar (SEA) was also used with a composition of 200 ml of soil and 1 liter of distilled water, heated at 200 o C on a hot stir plate for 1.5 hours.The soil solution was then filtered and added by 17 grams of pure microbiological agar and 0.1 grams of chloramphenicol.

Isolation of P. capsici on pepper (Piper nigrum) leaves.
Isolation was carried out on samples of pepper leaves showing symptoms of the disease.The leaf samples were rinsed using running water for 5 minutes, then the leaves with symptoms of the disease were cut from the healthy part with a size of ± 1 cm2.Surface sterilization used 1% NaOCl solution for ± 2 minutes and it was washed with distilled water three times, each for 1 minute.The sterilized leaf samples were then inoculated on CMA and PDA media directly in a petri dish, each containing 5 leaf pieces.They were then incubated at room temperature.The results of isolated leaf pieces showing the characteristics of the mycelium of P. capsici were immediately purified on PDA [6].

Morphological and molecular identification of P. capsici)
Fungus identification was carried out by macroscopic and microscopic observations.Microscopic observation was carried out by making semi-permanent preparations.The mycelium from the fungus was placed on top of the glass object.One drop of lacto phenol cotton blue solution was spread evenly, then left for a few minutes and covered using a glass cover.The microscopic identification used Phyopthora Diseases Worldwide identification key [7].Observation of hyphae microscopically was carried out by assessing the structure of the hyphae (septate or non-septate and hyaline or colored), shapes of asexual and sexual reproduction, as well as the structure of the fruit bodies.
Molecular identification was carried out by DNA isolation and PCR was carried out by sending pure culture samples of P. capsici on CMA media to the ICBB (International Culture Center for Biodiversity and Biotechnology) Molecular Analysis Institute.PCR products were sent to the paying agency First-BASE Laboratories, Malaysia for sequencing.The sequencing results were analyzed using the multiple alignment program, Clustal W with Bioedit 7.2.5 software, and aligned with the published nucleotide sequences on the GenBank website using the BLAST-N (Basic Local Alignment Search Tool-Nucleotides) program on the NCBI (National Center for Biotechnology Information) website.The homology value in each sample was determined using Clustal X 2.
2.5.Mycelium growth of P. capsici on various media.Mycelium growth of P. capsici was observed by direct inoculation of mycelium on seven media, including PDA, MA, CA, WA, CMA SEA, and V8.Mycelium diameter was measured after two days of inoculation.Measurements were made up to the 10th day after inoculation.Then, each mycelium colony of P. capsici from each medium was stored at room temperature for 40 days.This aims to see the ability of the mycelium to grow again after being stored in the [8][8] media for 40 days.After 40 days of storage, the diameter of the mycelium of P. capsici was measured again for 6 days to determine the effectiveness of the seven media on mycelium growth.2.6.Mycelium growth of P. capsici on various media.2.6.1.Production of P. capsici zoospores.The formation of P. capsici zoospores follows the method of [8] which has been modified.P. capsici pieces of 0.5 m 2 were grown in the V8 medium and incubated at 25 o C in the dark for 8 days until the media was filled with mycelia.The growing mycelium of P. capsici was then cut into square shapes with a size of 0.5 cm 2 .The P. capsici pieces were made into two parts and placed in a petri dish containing 10 ml of sterile water.They were then incubated for one hour at room temperature.The P. capsici isolate soaked in water was replaced with 20 ml of sterile water, incubated for 24 hours at room temperature with light, then incubated again at room temperature for 1 hour.Calculation of spore density was carried out using a haemacytometer.

2.6.2.
Virulence of P. capsici zoospores on pepper leaves.Observation of the virulence of P. capsici was carried out by taking 0.5 cm of P. capsici isolates grown on PDA, MA, CA, WA, CMA, SEA, and V8.P. capsici inoculation was carried out by taking isolates on the original media prior to storage and P. capsici which had been stored for 40 days.The effect of virulence of P. capsici on pepper leaves was determined by inoculating 0.5 cm of mycelium on pepper leaves stored in petri dishes filled with sterile tissue to keep them moist.Measurement of the diameter of the symptom area formed and calculation of the symptom area was carried out using the Image application, which can display the symptom area caused by P. capsici in cm 2 .

Isolation and Morphological Identification of P. capsici from Pepper Plants.
The results of the exploration of Foot Rot disease on pepper leaves in the field showed symptoms of Foot Rot on the leaves in the form of yellow leaf spots or chlorosis with a particular pattern, in which there is a jagged pattern at the ends of the spots, and when exposed to light, the spots will appear watery.In addition, there is a change in color at the base of the stem, with the initial symptoms being yellowish brown, then blackish brown, and finally, black and giving off a foul odor (Figure 1).Morphological identification was carried out by observing the mycelium characters of P. capsici on 7 different media.The character of the mycelium that appears on PDA media is white, resembling thick cotton, and has a rose pattern, whereas on CA, WA, and V8 media, the mycelium is white, resembling thick cotton, but not shaped like roses.On CMA and SEA media, mycelium growth showed a thin white color, not resembling cotton, and has a rose pattern (Table 1).White, has thin and cotton-like hyphae, the hyphae are not floral in shape, and do not form sporangia 40 days after incubation Water Agar (WA) White, has thin and cotton-like hyphae, the hyphae are not floral in shape, and do not form sporangia 40 days after incubation Corn Meal Agar (CMA) Yellowish white, has thin hyphae with hyphae motifs like roses, and produces pear-shaped sporangia.Soil Extract Agar (SEA) White, has thin hyphae, the hyphae are not floral in shape, and do not form sporangia 40 days after incubation Vegetable-8 (V8) White, has thick and cotton-like hyphae, the hyphae are not floral in shape, and do not form sporangia during the observation This study succeeded in isolating Foot Rot disease on pepper plants, in which the observation of P. capsici isolates from pepper has been characterized by its morphology on 7 growing media (Figure 1).Additionally, P. capsici isolates managed to be stored longer on SEA medium, able to maintain zoospore viability stability within 40 days of storage at 25 o C compared to using common growth media, such as CMA, PDA, and V8.SEA medium is able to maintain viability in producing spores because it is a medium that has better nutrient availability for P. capsici growth.Further, P. capsici is able to form chlamydospores as a survival phase and will reform zoosporangia when environmental conditions, such as temperature, light, and availability of nutrients are supportive.[1] explained that the ability of P. capsici to survive during the shelf life would form chlamydospores as survival structures that could survive the lack of nutrition and aeration during the storage period of 40 days.Chlamydospores will form a sporangium again if the availability of nutrients is abundant and environmental conditions support the development of P. capsici hyphae The results of observing the morphological form of P. capsici on various types of media showed that the morphological characteristics of hyphae and colonies before and after 40 days of storage did not change.
PDA and CMA media are good media for the growth of P. capsici colonies.[2] reported that PDA is the best medium for colony growth, the highest addition of biomass, and has spore germination of up to 95% in a short time.In contrast to the PDA medium, the growth of P. capsici colony mycelium in the MA medium is very low, so it is not recommended as a growing medium for P. capsici.This is because MA uses rose bengal as the media composition.The use of rose bengal is considered to inhibit the development of P. capsici hyphae.In addition, the growth of P. capsici in the MA medium was not able to produce zoospores before and after the shelf life.[9] reported that the use of rose bengal was able to inhibit the growth of mycelium from molds, while rose bengal was able to inhibit the growth of Candida albicans with an inhibition rate of 88.6±3.4% [10].DNA amplification of P. capsici using primers ITS 1 and ITS 4 was successfully amplified with DNA band of 836 bp (Figure 3).The BLAST results showed that isolate number 1 had a 99.75% similarity with P. capsici.

Mycelium growth of P. capsici on various media. 3.3.1. Mycelium growth of P. capsici after inoculation on various media.
The mycelium growth of P. capsici on 7 types of media was observed based on the diameter of the mycelium on the 2nd, 4th, 6th, 8th, and 10th day after incubation (hsi) (Table 2).

Mycelium viability of P. capsici after 40 days of storage
The The mycelium viability of P. capsici was determined through storage on media for 40 days.The mycelium viability was indicated by the increase in the size of the mycelium diameter which was observed on the 2 nd , 4 th , 6 th , and 8 th day after 40 days of storage (Table 2).Of the 7 media used to determine the mycelium viability of the P. capsici, only two media did not show the ability of the mycelium to continue growing after the storage period, including MA and WA media.This is presumably because the composition of the MA and WA media cannot support the mycelium viability for a long time, so the mycelium is unable to grow again due to the minimal nutrient content in the media.
Viability can also be observed based on mycelium growth rate (cm/day).P. capsici isolates grown on CMA medium had the highest growth rate of 0.15 cm/day with a mycelium diameter of 8.00 cm after 10 days of incubation at room temperature.Meanwhile, the mycelium growth rate in the PDA medium was 0.12 cm/day, and the growth rate in the V8 medium was 0.13 cm/day.Each medium showed an identical diameter on the 8th day after incubation, which was 8.00 cm.
Based on these results, it can be seen that the mycelium growth rate of P. capsici in the CMA medium is the best when compared to that of PDA and V8 media.Thus, PDA, CMA, and V8 media are the three types of media that best support the mycelium viability of P. capsici.However, based on the results of statistical analysis, the mycelium diameter and growth rate were not significantly different in each of the media used.The results of this study support the research conducted by [2] which stated that PDA and CMA were recommended media for the growth of P. capsici from chili plants.
In general, the production of zoospores on 7 types of media after soaking in sterile distilled water and incubating for 18-24 hours showed a significant difference in the number of zoospores (P≤0.05).SEA medium was able to produce higher zoospores, particularly 5.42x106/ml compared to the treatment on PDA, CMA, CA, and V8 media.This is in line with the symptom area on pepper leaves before the storage period, which was 18.61 cm2 and after the storage period, which was 16.69 cm2.Thus, the high virulence and viability of P. capsici in causing disease are indicated by the relatively high area of symptoms.In this study, hyphae from P. capsici isolates were able to cause necrosis symptoms on pepper leaves although some of them did not produce zoospores.This condition is caused by hyphae which are part of an infective plant pathogen known as a propagule.[11] reported that to infect a plant, a pathogen must have sufficient propagules.(SEA), (g) Vegetable-8 (V8).The results of this study show that P. capsici grown on five media (PDA, SEA, CMA, CA, and V8) can be purified again and has the ability to pathogenicity, zoospore production, and high viability as indicated by its ability to re-infect pepper leaves with a symptom area which remains quite wide, ranging from 15.01 cm2 to 21.85 cm2.Furthermore, the use of the five types of media as artificial media for P. capsici did not reduce the pathogenicity, zoospore production, and viability of P. capsici which had been stored for 40 days.[2] reported that PDA, CMA, and WA media were generally used as growth media, sporangia production/germination, and biomass increase from P. capsici.Not all inoculums in the soil can cause disease in plants because of their ability to form resistant structures and the aggressiveness of propagules as well as their survival which is different from that of P. capsici inoculums [7].
The mycelium growth of P. capsici on soil extract agar (SEA) medium was able to maintain a high number of zoospores after 40 days of incubation at room temperature.Soil extract agar medium is the best storage medium for P. capsici compared to other media.The slow growth on SEA medium indicated that the mycelium of P. capsici was still growing at a slow rate, in contrast to the growth of the mycelium of P. capsici on PDA, CMA, CA, and V8 media which stopped growing after 40 days of incubation.This is presumably because the soil extract agar medium contains minerals and nutrients that P. capsici can utilize as a source of nutrition [12] The nutrients required by microorganisms for growth include carbon, nitrogen, non-metal elements, such as sulfur and phosphorus, metal elements such as Ca, Zn, Na, K, Cu, Mn, Mg, and Fe, vitamins, water, and energy [13].
The viability of P. capsici inoculum sources before the storage period showed normal hyphal growth after being stored at 25 o C for 40 days and then purified.The results of growth observations in PDA, SEA, CMA, CA, and V8 media showed that there was no inhibition of zoospore production and it did not reduce the pathogenicity of P. capsici so the survival ability of P. capsici could last for a longer time during storage.The high virulence and pathogenicity characteristics are demonstrated by the ability of P. capsici to cause BSR symptoms which have been tested on pepper leaf pieces in sterile petri dishes.BSR symptoms on pepper leaves indicated necrosis in the P. capsici inoculation area, in which the symptoms appeared 48 hours after incubation at room temperature.Research results by [4]showed that there were 5 P. palmivora isolates that had high virulence even though they had been stored for 6-8 years in Sterile Distilled Water (SDW) medium (Table 4).SEA medium is the best medium for long-term storage of P. capsici.This is indicated by the hyphae conditions at the start of purification which remain in good condition for virulence and pathogenicity before and after the shelf life.Meanwhile, CMA, PDA, and V8 media were the best media for the growth of P. capsici colony mycelium.SEA, CMA, CA, V8, and PDA media did not inhibit hyphal development, zoospore production, and pathogenicity of P. capsici.However, the use of the PDA medium is still not able to maintain well in forming zoospores.[14] reported that viability is highly dependent on the quality of the inoculum source before being stored for a certain period 4. Conclusions P. capsici is able to survive in SEA medium for up to 40 days after incubation.Storage of P. capsici in the SEA medium did not decrease zoospore production, viability, virulence, and pathogenicity.The best artificial media to observe the form of colonies and hyphal growth of P. capsici are CMA, PDA, and V8 media.This study facilitates the determination of the type of artificial media that is effective in increasing the viability, zoospore production, and pathogenicity of P. capsici isolates after being stored at 25 o C.

Figure 3 .
Figure 3. Visualization of P. capsici PCR products on electrophoresis with 1 kb markers..

Figure 4 .
Figure 4. Viability of P. capsici after a shelf life of 40 days on media, (a) PDA, (b) Martin Agar (MA), (c) Carrot Agar (CA), (d) Water Agar (WA), (e) Corn Meal Agar (CMA), (f) Soil Extract Agar(SEA), (g) Vegetable-8 (V8).The results of this study show that P. capsici grown on five media (PDA, SEA, CMA, CA, and V8) can be purified again and has the ability to pathogenicity, zoospore production, and high viability as indicated by its ability to re-infect pepper leaves with a symptom area which remains quite wide, ranging from 15.01 cm2 to 21.85 cm2.Furthermore, the use of the five types of media as artificial media for P. capsici did not reduce the pathogenicity, zoospore production, and viability of P. capsici which had been stored for 40 days.[2] reported that PDA, CMA, and WA media were generally used as growth media, sporangia production/germination, and biomass increase from P. capsici.

Table 1 .
Description of P. capsici colony characters on various types of media.

Table 2 .
Effect of media on the mycelium growth of P. capsici before storage.

Table 3 .
Mycelium viability of P. capsici on various media.

Table 4 .
Zoospore production and virulence were observed from the area of symptoms of P. capsici after being stored on various media.