Sensitivity of Different Isolates of Pythium aphanidermatum to Old and Novel Fungicides

Among different groups of cultivated plant species, vegetables are the most sensitive to soil-borne pathogens due to their tender nature. They are frequently affected by many destructive diseases such as damping-off, wilt, and root rot, which are caused by species of genera Fusarium, Pythium, Phytophthora, Verticillium, Macrophomina, and Rhizoctonia (Koike et al., 2003; Noble and Coventry, 2005; Persley et al., 2010). The species of Pythium (class Oomycetes, order Pythiales) are not true fungi and, therefore, are classified in a separate kingdom Straminopila (Alexopoulos et al., 1996; Dick et al., 1999). The family Pythiaceae contains the two most economically important genera, Phytophthora and Pythium, each consisting of more than 200 species (Hyde et al., 2014; Brasier et al., 2022). Their species are considered the most damaging phytopathogens, causing huge economic losses worldwide (van West et al., 2003; Alexopoulos et al., 1996). Within the genus Pythium, P. aphanidermatum (Edson) Fitzpatrick is distributed worldwide but more common in warmer regions. It has a wide host range, infecting vegetables, ornamentals, fruits, and field crops (Lodhi, 2007; Waterhouse and Waterston, 1964; ; ). It caused different diseases in crop plants, varying from host to host, including seedling mortality, root rot, damping-off, stalk rot, cottony leak, and blight. Moreover, new hosts and diseases caused by this destructive pathogen are continuously reported elsewhere (, ). In severe cases of damping-off and seedling rots, total crop loss can happen (). The number of factors including, rapid growth and fast multiplication, soil-borne, aquatic nature, and tolerance to high temperature makes the control of P. aphanidermatum difficult. In order to suppress its infection, various control measures, including synthetic fungicides, plant extracts, biocontrol agents, and cultural practices, were tested (; ; ). Fungicides provide quick and effective control and most farmers depend upon them to get rid of such destructive pathogens. Since the introduction of the Bordeaux mixture in the late nineteenth century, many fungicides were developed for the control of destructive plant pathogens. Agrochemical sectors have been continuously in search of novel compounds that will be more effective, less toxic, and environmentally friendly. Accordingly, these fungicides were tested against different pathogens under in vitro as well as in vivo conditions (; ; ). However, over time, fungicidal resistance has been developed in some pathogenic species. Moreover, within the single species isolates of different origins would have different levels of fungicidal sensitivity (; ; ). The present study was also planned to screen out all the novel fungicides available against different isolates of Pythium aphanidermatum isolated from various vegetables grown in Sindh, Pakistan.

Roots and rhizospheric soil samples of different vegetables were collected from different locations in district Hyderabad. The samples were labeled and brought to the laboratory for isolation. For isolation of the P. aphanidermatum, a slightly modified baiting technique (Harvey, 1925) was used. Soil paste was made by adding distilled sterilized water (DSW), one teaspoon of this paste was placed on one side of sterilized Petri plates, added 10-15 ml of DSW in plates without disturbing the soil. Further, 2-3 sterilized grass blades of 2-3 cm long were also placed on each Petri plate, one near the soil and the other opposite side of the soil. These plates were kept at room temperature (25-30°C). After 5-7 days these grass blades having some mycelial growth at edges were washed with DSW and placed in new Petri plates followed by the addition of fresh DSW and grass blades. After 2-3 days of incubation, these grass blades were transferred to PDA plates containing Nystatin @100,000 L-1 to check the undesired fungi. The emerging colonies were purified by subsequent hyphal tip transfer on fresh agar plates. The targeted pathogen was then identified based on sporangial and oogonial characters (Plaats-Niterink, 1981). Finally, four isolates of P. aphanidermatum isolated from different sources were maintained on agar medium for further studies.

Different commercial fungicides (Table 1) available in the market were evaluated against P. aphanidermatum by the poisoned food method (Singh and Milne, 1974). A wide range of concentrations of these fungicides viz., 250, 500, 1000, 2000, 4000, 6000, and 8000 ppm was prepared and mixed in PDA before sterilization. PDA without fungicides served as control. After solidification of the medium, a 5 mm disc from 7 days old culture was placed in the center of the Petri plate. Similarly, the response of four selected isolates was evaluated against each fungicide and their diffident concentrations individually. These plates were incubated at 30°C till the control plates were

Table 1: Details of fungicides used against Pythium aphanidermatum.

fully covered with the growth of P. aphanidermatum. The experiment was arranged completely randomized design (CRD) with six replications. The percent inhibition of fungus was calculated by using the formula given below:

PIRG = (R1 – R2)/R1 x 100 (Vincent, 1947)

Where; R1: Colony growth of pathogen in control plate (without fungicide). R2: Colony growth of pathogen in the treated plate (with fungicide).

The Statistix 8.1 software was used to calculate the analysis of variance (ANOVA). Mean values were compared by least significant difference (LSD) test at p< 0.05 (Statistix, 2006).

The fungicides main effect (DF= 16, F= 339741, P= 0.00), concentration’s main effect (DF= 6, F= 112554, P= 0.00) and fungicides × concentration’s effect (DF= 96, F= 19665.0, P= 0.00) are highly significant (Table 2). Among 17 fungicides, Copper oxychloride, Score, Tilt, Amistar Top, Nativo, Contaf Plus, Ridomil Gold, Systhane, and Moncut appeared highly effective, but the tested isolate failed to grow at all concentrations, even at 250 ppm. In contrast, fungicides like Revus and Evito appeared effective, their highest concentration i.e., 8000 ppm completely failed to check the growth of Pa 11. The remaining fungicides were ineffective at lower concentrations but became highly effective at higher concentrations. The 250-1000 ppm of Alliete, Antracol, and Melody Duo completely failed to inhibit the growth of the test pathogen, while higher concentrations (4000-8000 ppm) checked the pathogen growth. In the case of Dynasty, the pathogen growth was completely inhibited by all concentrations except 250 ppm, which completely failed to inhibit the growth. In the case of Curzate, the pathogen successfully grew at 250-4000 ppm but was completely inhibited at 6000 and 8000 ppm. In the case of Topsin M, the growth of Pa 11 gradually reduced with increasing concentrations from 250 to 2000 ppm and completely stopped at 4000 to 8000 ppm (Table 3).

Table 2: Analysis of variance of fungicides and concentrations for P. aphanidermatum 11.

Table 3: Response of P. aphanidermatum 11 to different concentrations of various fungicides.

Each value is a mean of six replications. Values with different letters differ significantly at p < 0.05 as determined by DMRT.

Table 4: Response of P. aphanidermatum 12 to different concentrations of various fungicides.

* Each value is a mean of six replications. Values with different letter differ significantly at p < 0.05 as determined by DMRT.

There was a great variation in the efficacy of different fungicides against Pa 12. Two fungicides i.e., Revus and Evito failed to cause any negative effects on colony growth of Pa 12, the highest used concentration of these fungicides (8000 ppm) produced the growth identical to the control treatment (w/o fungicide). Pa 12, showed tolerance to 250-1000 ppm of Alliete, in which test pathogen grew vigorously, but completely inhibited at 2000-8000 ppm. In Curzate, 250 and 500 ppm failed to produce any inhibition, while, 1000, 2000, and 4000 produced 44.44, 70.37, and 77.78% inhibition, respectively, and completely inhibition at 6000 and 8000 ppm. In Antracol, 250 and 500 ppm produced no inhibition, 1000 ppm caused 33.33% inhibition and 2000-8000 ppm caused 100% inhibition. The 250, 500, and 1000 ppm of Melody Duo failed to cause any reduction, 2000-8000 ppm caused 77.78-100% gradual inhibition in the colony growth of the test pathogen. Other remaining fungicides viz., Dynasty, Copper oxychloride, Score, Tilt, Amistar Top, Nativo, Contaf Plus, Ridomil Gold, Systhane, and Monocot were found highly effective, and their all concentrations produced 100% inhibition (Table 4). On an overall basis, fungicides showed highly significant impact (DF= 16, F= 341178, P= 0.00). The fungicide concentration’s effect was also highly significant (DF= 6, F= 131659, P= 0.00). The interactive effect of fungicides and concentration also appeared highly significant (DF= 96, F= 21200.2, P= 0.00) (Table 5).

Table 5: Analysis of variance of fungicides and concentrations for P. aphanidermatum 12.

In terms of effectiveness against Pa 14, the 17 fungicides were divided into three groups i.e., highly effective fungicides at all concentrations, moderately effective-not effective at lower concentrations but effective at higher concentrations, not effective at all concentrations. The overall impact of fungicides was highly significant (DF= 16, F= 602459, P= 0.00). The overall response of concentrations to pathogen inhibition was also highly significant (p < 0.001). The ‘fungicides × concentrations’ to inhibition percentage also found highly significant (DF= 96, F= 41491.6, P= 0.00) (Table 6). The first group comprises Dynasty, Copper oxychloride, Score, Tilt, Amistar Top, Nativo, Contaf Plus, Ridomil Gold, Systhane, and Monocot, the pathogen failed to grow at all tested concentrations of these fungicides. The second group consists of Alliete, Curzate, Antracol, Melody Duo, and Topsin M, which appeared completely or partially ineffective at 250-1000 ppm, but completely inhibited the pathogen growth at 2000-8000 ppm. The third group consists of Revus and Evito, which completely failed to check the growth of Pa 14 at all concentrations (Table 7).

Table 6: Analysis of variance of fungicides and concentrations for P. aphanidermatum 14.

Analysis of variance showed that for pathogen inhibition, there is a highly significant difference between fungicides (DF=16, F=360762, P=0.000), among concentrations (DF= 6, F= 133770, P= 0.000) and the interactive response of fungicides and concentrations (DF= 96, F= 22555.4, P= 0.000) (Table 8). The response of Pa 16 to fungicides to almost similar to those of Pa 14. Among 17 fungicides, Revus and Evito were found to be ineffective; the higher concentration (8000 ppm) produced the same growth of pathogen that recorded in the control (w/o fungicides). On the other hand, 10 fungicides (Dynasty, Copper oxychloride, Score, Tilt, Amistar Top, Nativo, Contaf Plus, Ridomil Gold, Systhane, and Monocot) appeared highly effective, the test isolated failed to grow even at 250 ppm. The remaining five fungicides showed a mixed response to the test pathogen.

Table 7: Response of P. aphanidermatum 14 to different concentrations of various fungicides.

Each value is a mean of six replications. Values with different letter differ significantly at p < 0.05 as determined by DMRT.

Table 8: Analysis of variance of fungicides and concentrations for P. aphanidermatum 16.

Alliete became completely ineffective at 250, 500, and 1000 ppm, but highly effective at 2000 ppm and additional concentrations. Similarly, no inhibition occurred at 250-1000 ppm of curzate, 2000 ppm produced 55.56% inhibition; 4000 ppm produced 77.79%, and no growth at 6000 and 8000 ppm. Antracol also successfully inhibited complete growth at 4000-8000 ppm, melody duo and topsin M brought 100% inhibition at concentrations of 2000-8000 ppm (Table 9).

All isolates of P. aphanidermatum were successfully grown when exposed to different concentrations of Revus and Evito. Both fungicides completely failed to cause any negative effects on in vitro growth of all the four isolates. Their growth rate at a very higher concentration of 8000 ppm was almost equal to those of control (w/o fungicides). In contrast to Revus and Evito, all isolates appeared extremely sensitive to 10 fungicides including Dynasty, Copper oxychloride, Score, Tilt, Amistar Top, Nativo, Contaf Plus, Ridomil Gold, Systhane, and Monocot. All isolates except Pa 12 (which grew only at 250 ppm of Dynasty) failed to tolerate 250-8000 ppm concentrations of these fungicides and produced no growth under in vitro conditions. To some extent, the tested isolates showed variable responses to Alliete, Melody Duo, Topsin M, Curzate, and Antracol. In the case of Alliete, all isolates grew well at 250-1000 ppm and completely stopped growing at 2000-8000 ppm, except Pa 11 which produced moderate growth at 2000 ppm. In the case of Melody Duo, Pa 11 and Pa 12 showed tolerance up to 4000 ppm; while, Pa 14 and Pa 16 grew up to 1000 ppm. In the case of Topsin M, Pa 11 and Pa 12, grew up to 2000 ppm, but Pa 14 and Pa 16 only grew up to 1000 ppm. Such type of variable response of four isolates was also found in the case of Curzate and Antracol (Table 10).

Table 9: Response of P. aphanidermatum 16 to different concentrations of various fungicides.

Each value is a mean of six replications. Values with different letter differ significantly at p < 0.05 as determined by DMRT.

Table 10: Comparative sensitivity of different isolates of P. aphanidermatum to different fungicides.

Each value is a mean of six replications. Values with different letter differ significantly at p < 0.05 as determined by DMRT.

Use of fungicide for commercial seeds treatment of various crops for the management of soil-borne diseases such as caused Pythium spp. increasingly become crucial (Doshi and Mathur, 1987; Mocioni et al., 2003; Gerik, 2005; Ahmadzadeh and Sharifi-Tehrani, 2006; Carmona et al., 2018; Lookabaugh et al., 2021; Doherty and Roberts, 2022). Metalaxyl is being used successfully for its management. However, the rise of Pythium-resistant strains resistant to widely used Metalaxyl-M has emphasized the need for other technologies to fungicides for disease control (Cohen et al., 1999). Discovery and evaluation of alternative novel compounds that are more effective, less toxic, and environmentally friendly are highly desirable. The developed compound needs to check against the available isolates of candidate species. With this aim present study deal with the in vitro evaluation of 17 systemic and non-systemic fungicides against 4 isolates of the prominent Pythium species (P. aphanidermatum) causing diseases to many crops. All isolates appeared extremely sensitive to Dynasty (azoxystrobin), Copper oxychloride (copper oxychloride), Score (difenoconazole), Tilt (propiconazole), Amistar Top (azoxystrobin and difenoconazole), Nativo (trifloxystrobin and tebuconazole), Contaf Plus (hexaconazole), Ridomil Gold (mancozeb and mefenoxam), Systane (myclobutanil) and Monocot (flutolanil), Alliete (fosetyl aluminium), Melody Duo (iprovalicarb and propineb), Topsin M (thiophanate methyl), Curzate (cymoxanil and mancozeb) and Antracol (propineb). Several studies focus on the evaluation of different fungicides against P. aphanidermatum. Such studies are important as all the effective fungicides could be used alternatively to reduce the development of fungicide resistance (Suleiman, 2011). Azoxystrobin and mefenoxam were sensitive to Pythium spp., (Matic et al., 2019). The fungicides Benlate (benomyl), Ridomil, and mancozeb at 50-200 ppm significantly reduced in vitro mycelial growth of P. aphanidermatum (Suleiman, 2011). For P. aphanidermatum and P. deliense, Ridomil, Galben M, Warmine, Orthocide + Difolatan and Teroxin effectively reduced the growth of colony at low concentrations (Singburaudom et al., 1981). Ethaboxam (benzamide) anti-oomycete chemical (Noel et al., 2019). Phosphonic acid-based fungicides (phosphonates) are efficient versus diseases caused by oomycetes (Dann and McLeod, 2021). Fluazinam also was effective in the inhibition of P. aphanidermatum (Cohen et al., 1999).

Sensitivity to fungicides also varied from species to species within the genus Pythium as well as within the different isolates of the same species. Against 21 isolates of P. aphanidermatum, the Ec50 of metalaxyl was ranging from 1.19 to 3.12 μg ml-1 and 0.05 to 1.30 μg ml-1 for P. ultimum (Brantner and Windels, 1998). In the present investigation, different P. aphanidermatum isolates showed variable growth responses to different used doses. All isolates except Pa 12 appeared resistant against 250-8000 ppm concentrations of these fungicides except dynasty and produced no growth under in vitro conditions. different isolates may exhibit variation in fungicidal sensitivity because of different origins. A study conducted in Oman indicated that out of 27, only one isolate of P. aphanidermatum was found resistant to hymexazole (Al-Balushi et al., 2018). Mostly the isolates of the same origin have less variation against fungicides, but differences to fungicides become more visible if a large and diverse population were tested (Al-Sa’di et al., 2008). Discussing the same point Matic et al. (2019) observed that azoxystrobin was equally effective against different isolates of six Pythium spp., with no azoxystrobin-resistant isolate. However, the same complex showed variable response to mefenoxam. Moreover, some of these isolates showed resistance to mefenoxam as well. In another study, out of 194 isolates belonging to four genera of peronosporalean lineage, 16% were insensitive to Ethaboxam (benzamide). Discovering the mechanism of insensitivity, they found mutations in the 239th codon in beta-tubulin as benzamides are hypothesized to bind to beta-tubulin (Noel et al., 2019).

A great variation in fungicides efficacy has been observed. Not all but only specific fungicides are effective against P. aphanidermatum. Different P. aphanidermatum isolates exhibited slight variation in fungicidal sensitivity. In areas of hot spots, the infection of P. aphanidermatum can be controlled by the application of azoxystrobin, copper oxychloride, difenoconazole, propiconazole, azoxystrobin + difenoconazole, trifloxystrobin + tebuconazole, hexaconazole, mancozeb + mefenoxam, myclobutanil and flutolanil. The present study also reveals a slight variation among P. aphanidermatum population, therefore, either varietal evaluation studies and/or testing different control measures presence of distinct isolates should be considered.

The present study reveals the response of four different isolates of destructive and widespread soil-borne pathogens Pythium aphanidermatum to 17 old and novel synthetic fungicides.

Ghulam Sarwar Shah: Investigation and data curation and drafted part of it.

Maqsood Anwar Rustamani and Rab Dino Khuhro: Reviewed, editing, and approved the manuscript.

Rehana Naz Syed: Analyzed the data and drafted part of the manuscript.

Abdul Mubeen Lodhi: Analyzed, edited and approved the manuscript.

All authors have read and agreed to the published version of the manuscript.

The authors have declared no conflict of interest.