Next Article in Journal
Perceived Social Impacts of Protected Areas, Their Influence on Local Public Support and Their Distribution across Social Groups: Evidence from the Eifel National Park, Germany, during the COVID-19 Pandemic
Previous Article in Journal
The Spatial Transformation of the Villages around Chang’an Cultural Heritage Site Based on Actor Network Theory
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Sustainability
Volume 15
Issue 14
10.3390/su151410842
sustainability-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Vernonia amygdalina Extract Loaded Microspheres for Controlling Phytophthora palmivora

by
Jiratchaya Chainanta
1,
Kittiporn Yakajay
2,
Chanakarn Chinda
2,
Phikul Intaparn
1,
Chaiwat To-anun
1,
Pratchaya Tipduangta
2,
Busaban Sirithunyalug
2,
Sukanya Haituk
1,
Jeerapa Nguanhom
3,
Tonapha Pusadee
4,
Anuruddha Karunarathna
1,* and
Ratchadawan Cheewangkoon
1
1
Department of Entomology and Plant Pathology, Faculty of Agriculture, Chiang Mai University, Chiang Mai 50200, Thailand
2
Department of Pharmaceutical Sciences, Faculty of Pharmacy, Chiang Mai University, Chiang Mai 50200, Thailand
3
Faculty of Agricultural Technology, Chiang Mai Rajabhat University, Chiang Mai 50200, Thailand
4
Department of Plant and Soil Science, Faculty of Agriculture, Chiang Mai University, Chiang Mai 50200, Thailand
*
Author to whom correspondence should be addressed.
Sustainability 2023, 15(14), 10842; https://doi.org/10.3390/su151410842
Submission received: 24 April 2023 / Revised: 30 June 2023 / Accepted: 3 July 2023 / Published: 11 July 2023
(This article belongs to the Special Issue Plant-Microbe Interactions for Sustainable Agriculture in Changing Environment)
Browse Figures
Versions Notes

Abstract

:
The adverse effects of modern fungicide consumption have caused many issues in the agroecosystem. Hence, under sustainable agriculture concepts, it is important to research alternatives to the currently used fungicide. The use of secondary metabolite-containing herbal extracts for treating plant diseases has become the latest trend in sustainable and green agriculture. However, the poor solubility and volatile nature of many compounds cause practical issues when using them in the field. Hence, bioactive compound delivery through nano- or micro-particles has become a successful technique to improve the solubility and delivery of secondary metabolites to targeted sites. In the current study, the ethyl acetate (EtOAc) extract from dried leaves of Vernonia amygdalina was tested against Phytophthora palmivora isolated from Durian (Durio zibethinus) root rot. Further, the potential of enhancing the effect of V. amygdalina EtOAc treatment through microspheres loaded with V. amygdalina EtOAc extract was also investigated. The microspheres encapsulated with Eudragit® E were synthesized under different electrospray conditions to obtain the microspheres with the highest efficacy. The poison media assays evaluated the fungal growth inhibition efficiency of the V. amygdalina EtOAc crude extract and the synthesized microspheres. The study reveals that the V. amygdalina EtOAc extract has the potential to suppress the growth of P. palmivora. Interestingly, the synthesized microspheres showed immense growth inhibition in P. palmivora, with a 61.10 µg/mL decrease in ED50 compared to the direct usage of V. amygdalina EtOAc extract.

1. Introduction

Durian (Durio zibethinus Murray; family Bombacaceae) is an important fruit crop for Thailand, covering 95% of the world’s Durian supply [1]. Oomycetes cause some prominent diseases in Durian, such as root rot, stem rot, and fruit rot [1]. However, these diseases are caused by several Oomycetes species, viz. Phytophthora palmivora, P. nicotianae, and Pythium cucurbitacearum. Durian root rot caused by P. palmivora is a serious disease in Thailand [2]. The majority (72.8%) of Durian cultivation in Thailand is affected by the root rot disease [3]. The soil-borne P. palmivora initially affects the plant roots and gradually causes the death of the whole plant [4]. A broad range of chemicals, such as etridiazole, fosetyl-Al, phosphonic, and metalaxyl, are used to control the disease [5,6]. However, the excessive and prolonged use of chemicals causes ecological issues, higher production costs, and fungal resistance to chemical fungicides [1]. Several countries, such as the United Kingdom [7], Cameroon [8], China [9], Estonia [10], Mexico [11,12], Morocco [13], Poland [14], Russia [15], Uganda [16], and the United States [17,18,19,20], have reported fungicide resistance to Phytophthora spp. Hence, proper identification as well as identifying the proper treatment strategies are important for disease control.
Sustainable agriculture demands efficient production, safe resource consumption, resource conservation, protection of the farm ecosystem, and protection and improvement of the natural environment, along with safeguarding the social and economic conditions of the farming communities [21]. However, current agriculture practices with prolonged and excessive usage of fungicides cause irreversible damage to ecosystems [22]. Hence, the use of bioactive secondary metabolites for controlling diseases became a major topic in green and sustainable agriculture [23,24]. The major issues with using bioactive plant extracts for controlling fungal pathogens include less water solubility, less permeability, and the physicochemical instability of bioactive compounds in plant extracts [25,26]. Hence, green agriculture requires efficient techniques to deliver such compounds to the targeted site and activate the action. Modern technology in the synthesis of nanoparticles, microspheres, and micelles successfully addresses this issue [27,28,29,30]. Yen et al. [31] successfully demonstrated enhanced solubility and dissolution mechanisms of curcumin through the synthesis of polymeric curcumin nanoparticles. Chaetomium brasiliense extracts loaded with nanoparticles efficiently inhibit the mycelial growth and spore production of Phytophthora palmivora [32]. Further, the extracts of Chaetomium sp. loaded nanoparticles showed significant activity against Magnaporthe oryzae [33].
The formation of compound loaded nanoparticles has several downfalls, such as formulation complexities, lack of scalability, and cost [34]. However, Malik et al. [34] addressed the above shortcomings of nanoparticle production through the electrospray process. Malik et al. [34] were able to synthesize highly porous (>94%) microspheres while maintaining control over particle structure and size. Further, the process was highly reproducible. The electrospray technology for improving the solubility of less soluble Nintedanib (a new tyrosine kinase inhibitor and growth factor antagonist) through solid dispersion was successfully demonstrated by Liu et al. [35]. The molecular weight of the polymer used for microsphere production plays a major role in the encapsulation process of the active ingredient. Further, the polymer concentration affects the solution characteristics, such as pH, conductivity, viscosity, and surface tension. Hence, the polymer has a huge impact on the characteristics of the particles [36]. These parameters are well satisfied by Eudragit® E (dimethylamino ethyl methacrylate, butyl methacrylate, and methyl methacrylate-based cationic copolymer). Eudragit® E has been widely used as a polymer matrix for drug delivery [37]. Based on the aforementioned characteristics, we decided to use Eudragit® E for our tests.
Vernonia amygdalina Del (Nan Chao Wei) (Compositae) is a well-known medicinal plant in Asian and African traditional medicine [38]. All parts of the plant show antidiabetic, antioxidant, antimicrobial, anticancer, anti-inflammatory, and antiplasmodial effects [39]. Hence, many studies were conducted to research the ability to use V. amygdalina leaf extracts to control plant diseases [40,41]. The crude extract of V. amygdalina (leaves) loaded nanoparticles was successfully used to control Pythium deliense in Catharanthus roseus [40]. Further, the leaf extracts of V. amygdalina have been tested against Botrytis cinerea, the causative agent of gray mold disease on tomato fruits. Yusoff et al. [41] revealed the presence of a high fraction of antifungal compounds: squalene (16.92%), phytol (15.05%), triacontane (11.31%), heptacosane (7.14%), and neophytadiene (6.28%) in leaf extracts of V. amygdalina. Yusoff et al. [41] used the dichloromethane (DCM) extract of V. amygdalina for treating gray mold disease on tomato fruits. Dichloromethane extract of V. amygdalina affects the fungal morphology by shrinking and agglomerating the mycelia and conidia of Botrytis cinere, Yusoff et al. [41]. Later on, Yusoff et al. [42] developed a V. amygdalina leaf extract emulsion against Botrytis cinere. Further, Sesquiterpene lactones are known to be highly antifungal secondary metabolites [43,44]. Vernodalol and vernolide belonging to sesquiterpene lactones demonstrated higher antifungal effects against Penicillium notatum and Aspergillus flavus, with LC50 values of 0.4 mg/mL each [45]. Dar and Soytong [32] found that the C. brasiliense crude extract loaded with nanoparticles can effectively reduce P. palmivora colony growth and spore germination.
In the current study, a severe Durian root rot onset was observed in northern Thailand. The pathogen was identified as Phytophthora palmivora using Koch’s postulations, taxonomy, and phylogeny. Further, the efficiency of using V. amygdalina herbal extract loaded microspheres for controlling the Durian root rot caused by P. palmivora was investigated.

2. Materials and Methods

2.1. Selection and Identification of the Pathogen

2.1.1. The Sample Collection

Root and soil samples were collected from Durian orchards in Chiang Dao District, Mae Hia, and Mae Ai from August to December 2019. Both symptomatic and asymptomatic plants were selected randomly from the sample collection. All stages of the plants in the field were highly affected by visible dieback disease (Figure 1). The aerial crown of the trees showed extensive necrosis, starting from the tips to the base. The decline is slow. The leaves initially show symptoms similar to drought stress and eventually dry off. The lower part of the trunk, closer to the soil line, shows dark patches in the bark (Figure 1B,C). Brown discoloration has been observed under the necrotic bark pieces. The necrotic bark pieces are slightly moistened with exudate (Figure 1E). Water-soaked lesions are visible on the extensively affected root surface (Figure 1F). The dried-off outer layers of roots are easily peeled off, and the lightly colored inner parts are revealed (Figure 1G). The fine roots turned black and decayed. Symptomatic as well as asymptomatic roots were collected. Further, soil samples (500 g) surrounding the roots were collected from both symptomatic and asymptomatic plants. Samples were stored in plastic bags and transported to the laboratory within 24 h.

2.1.2. Isolation of the Pathogen

The pathogens were isolated from the samples using leaf baits [33]. For the baits preparation, healthy Durian leaves were washed with running tap water and patted dry using sterile tissue paper. Then the leaves were thoroughly washed with 75% ethyl alcohol, and the residual solution on the leaf surface was removed by air drying. The Leaves were cut into 5 mm × 5 mm pieces under sterile conditions. For pathogen isolation from soil samples, 5 g of soil was dispersed in sterile Petri dishes and flooded with sterile water. Ten leaf baits were added to the flooded Petri dishes and incubated for three days until the sporangia were visible in the bait margins. For the pathogen isolation from the root samples, the symptomatic and asymptomatic roots were selected separately. Roots were cleaned thoroughly in running tap water to remove the loosely bound debriefs. The roots were patted dry with sterile tissues and cut into 1 cm pieces. For each Petri dish, 20 pieces were transferred, and sterile water was added to fill up half of the Petri dish. Then 10 of the previously prepared leaf baits were added and incubated for three days until the sporangia were visible in the bait margins. Once the sporangia were visible on the baits from the soil and root samples, the baits were transferred into the sterile water under aseptic conditions. The baits were thoroughly washed. The washing was repeated three times, changing the water each time. After washing, the baits were placed on sterile filter papers until the excess water was dried off. Then the baits were transferred to 2% water agar (WA) plates with added antibiotics to suppress the bacterial growth. Four baits were transferred to each plate and incubated until the mycelia grew. Once the mycelia started to grow, hyphal tipping was performed, and the mycelial tips were transferred to Potato dextrose agar plates (PDA) with added antibiotics. Once the colonies were grown, the cultures were transferred to the PDA without antibiotics for further studies.

2.1.3. Pathogenicity Assay

All the isolates were tested for their pathogenicity using the detached leaf method and inoculated with mycelial plugs. Healthy leaves in between the young and mature developmental stages were used for the pathogenicity assay. Three leaves were used for each isolate. The leaves were washed thoroughly using running tap water for 1 min. Then the leaves were washed with 1% NaOCl for 1 min and washed with distilled sterile water for 1 min. The leaves were then washed with 70% ethyl alcohol for 1 min and three times washed with sterile distilled water for 1 min each time. The leaves were then patted dry using sterile tissue paper and kept in a moisture chamber. The mycelial plugs with a 5 mm diameter were cut from the growing ends of the five-day-old cultures on PDA. In each leaf, four wounds (two wounds on either side of the midrib) were made with a sterile needle. One mycelial plug was placed on each wound at the right-hand side of the midrib, and sterile 5 mm diameter PDA discs were placed on the wounds on the left-hand side of the midrib as the control. The disease’s appearance and progression were assessed every 12 h. Among the isolates, the most virulent isolate was selected for further studies.

2.1.4. Identification of the Pathogen

The genomic DNA of the most virulent strain was extracted using a commercial DNA extraction kit (FavorPrepTM Tissue Genomic DNA Extraction Mini Kit). The DNA amplification was performed by polymerase chain reaction (PCR) for the partial sequences of two gene regions: the internal transcribed spacers (ITS1, 5.8S, and ITS2), which were amplified using the primers ITS5 and ITS4 [46], and COX1, amplified using primer pairs OomCoxI- Levup and Fm85mod. Polymerase chain reaction (PCR) was carried out following the protocols described by Robideau et al. [47]. Phylogenetic analyses were conducted based on a combined gene of ITS and COX1 sequence data, following the methodology described by Wongwan et al. [48].

2.2. Plant Extraction

Vernonia amygdalina leaves (5 kg) were collected from Chiang Mai University, Mae Hia farm. The leaves were washed thoroughly with tap water and dried at room temperature. Dried leaves were placed in a tray and oven dried at 60 °C for 16 h. The dry leaves were powdered. The powder (680 g) was put in a cloth bag. The bag containing the powder was soaked in ethyl acetate (EtOAc) with a solid-to-solvent ratio of 1:5 w/v (680 g:3400 mL) and kept at room temperature for three days. The EtOAc layer was decanted, and a new portion of EtOAc with the same volume (3400 mL) was added. This step was repeated one more time, and the decanted EtOAc fractions were combined. The EtOAc fraction was filtered using filter papers (Whatman No. 4, Maidstone, UK). The solvent in the filtrate was removed using the rotary evaporator. The resultant dark green crude (41.11 g) was stored in an amber glass bottle at 4 °C until further use.

2.3. Phytophthora palmivora Inhibition Assay against V. amygdalina Crude Extract

The previously isolated P. palmivora isolate was transferred to a new PDA plate and incubated for five days. Mycelial plug disks (5.5 mm) were prepared from the growing end of the colony. The crude extract was dissolved in dimethylsulphoxide (DMSO) (0.2%). The resultant mixture was filtered using a Whatman™ Puradisc™ Nylon Syringe filter and added to PDA (40 °C) to make the concentration gradient: 0, 10, 50, 100, 500, and 1000 µg/mL. The PDA mixtures were then poured into Petri dishes (15 mL/plate). Previously prepared, mycelial plugs were transferred to poison PDA and incubated at room temperature. The results were recorded daily until the control reached full maturity by measuring the colony diameter to calculate the percent inhibition of radial growth (PIRG) and effective dose (ED50).

2.4. Producing a Crude V. amygdalina Extract into Particles

A custom-made electrospray apparatus was used in this study with a high voltage supplier, an ES40P-20 W power supply (Gamma High Voltage Research Inc., Ormond Beach, FL, USA), a syringe pump NE-1000 (New Era System), New Era Pump Systems, Wantagh, NY, USA), and a custom-made static collector covered with aluminum foil to collect the electrospray particles. The electrospray process was performed at ambient temperature and humidity (25–30 °C, 50–60% RH).

2.4.1. Electrospraying Process and Polymer Concentration Optimization

The parameters of the electrospraying were investigated at flow rates of 2 and 4 mL/h, voltages of 15 and 20 kV, and distances of 12 and 15 cm. The electrospray solution was prepared from 500 µg/mL of V. amygdalina crude extract (0.005 g) and 5% w/v Eudragit® E 100 (0.5 g) dissolved in EtOAc (10 mL). The effect of polymer concentrations was investigated on Eudragit® E 100 at 0.5, 1, 3, and 5% w/v with an electrospray parameter of 2 mL/h at 20 kV and a distance from the needle tip to the collector of 15 cm.

2.4.2. Electrospray Particle Morphology Study and Particle Size Measurement

The electrospray particles were examined under a JEOL JSM-7610F scanning electron microscope (SEM) (Tokyo, Japan). The diameter of the electrospray was measured using ImageJ software Version 1.51 (NIH, Bethesda, MD, USA) using Martin’s diameter [49]. Then, the histograms of the electrospray particles were plotted by ORIGIN 8.0.

2.5. Phytophthora palmivora Inhibition Assay against V. amygdalina Extract Loaded Microspheres

The previously isolated P. palmivora isolate was transferred to a new PDA plate and incubated for five days. Mycelial plug disks (5.5 mm) were prepared from the growing ends of the cultures. The microspheres loaded with V. amygdalina were dissolved in DMSO (0.2%). The resultant mixture was filtered using a Whatman™ Puradisc™ Nylon Syringe filter and added to PDA (40 °C) to make the concentration gradient: 0, 1, 5, 10, 50, and 100 µg/mL. The PDA mixtures were then poured into Petri dishes (15 mL/plate). Previously prepared, mycelial plugs were transferred to poison PDA and incubated at room temperature. The results were recorded daily until the control reached full maturity by measuring the colony diameter to calculate the percent inhibition of radial growth (PIRG) and effective dose (ED50).

3. Results

3.1. Selection and Identification of the Fungal Strain

A total of 30 isolates were obtained from soil (16 isolates) and root (14 isolates) samples (Table 1). Phytophthora spp. cultures on PDA were characterized by chrysanthemum-shaped colonies with entire margins, coenocytic hyphae, and spherical chlamydospores with thick walls (Figure 2).
Initially, the pathogenicity was tested for all 30 Phytophthora spp. isolates. Based on the results, Ph 410 showed the highest disease severity. The strain Ph 410 showed a circular succulent leaf spot in the first phase. On the fifth day, the spots showed necrosis from the center (1.8 cm) (Figure 3). The spot turned from brownish to gray. Our morphological observations of strain Ph 410 resemble the P. palmivora morphology described by Suzui et al. [50].
The RAxML analysis of the concatenated ITS and COX1 datasets yielded the best scoring tree (Figure 4), with a final ML optimization likelihood value of −7226.167050. The matrix had 484 distinct alignment patterns, with 6.11% of undetermined characters or gaps. The GTR+I+G model parameters of the combined ITS and COX1 were: Estimated base frequencies of A = 0.225914, C = 0.172480, G = 0.228260, T = 0.373346, and substitution rates of AC = 0.948393, AG = 3.235665, AT = 2.563264, CG = 0.946138, CT = 3.574835, and GT = 1.000000. Further, the proportion of invariable sites I = 0.378441 and the gamma distribution shape parameter α = 0.771308. Further, the ML phylogeny forms a strongly supported clade with P. palmivora. Hence, based on morphology and phylogeny, isolate Ph 410 was identified as P. palmivora (Figure 2 and Figure 4).

3.2. Phytophthora palmivora (Ph 410) Inhibition assay against V. amygdalina Crude Extract

The percentage inhibition of P. palmivora against the concentration gradient of 10, 50, 100, 500, and 1000 µg/mL was 18.44%, 31.6%, 47.78%, 72.22%, and 85.33%, respectively (Figure 5A). For the above concentrations, the calculated ED50 was 374.87 µg/mL (Table 2).

3.3. Optimization of Electrospray Microparticles Containing a Crude Extract from V. amygdalina

3.3.1. Electrospraying Process Optimization

The SEM image (Figure 6) shows that the feeding rate of 2 mL/h produced microspheres, while the feeding rate of 4 mL/h did not produce particles. Further, the 20 kV voltage produced particles with 6.6 ± 1.5 µm and 7.3 ± 3.1 µm under the two different collector and nozzle distances, 12 cm and 15 cm, respectively. The 20 kV voltage produced particles with 5.2 ± 1.7 µm and 5.5 ± 2.0 µm under the two different collector and gun distances, 12 cm and 15 cm, respectively. Based on the results, a feeding rate of 2 mL/h, a 20 kV voltage, and a 15 cm distance between the gun and the collector were used as the optimized conditions.

3.3.2. Polymer Concentration Optimization

Based on the SEM micrographs (Figure 7), Eudragit® E at 0.5% and 1% w/v formed aggregates. However, Eudragit® E at 3% and 5% w/v produced more spherical microspheres (4.1 ± 0.7 µm and 5.5 ± 2.0 µm, respectively) with dimples on them. Based on the results, the 3% w/v Eudragit® E was selected as it formed more even-shaped and even-sized microparticles (Figure 7 and Figure 8).

3.4. Phytophthora palmivora (Ph 410) Inhibition Assay against V. amygdalina Extract Loaded Microspheres

The Phytophthora palmivora inhibition percentages for the V. amygdalina extract loaded microsphere concentrations of 1, 5, 10, 50, and 100 µg/mL were 8.67%, 4%, 5.11%, 44.22%, and 89.56%, respectively (Figure 5B). The ED50 values for the above concentrations were 61.10 µg/mL (Table 2).

4. Discussion

Durio zibethinus is a highly important plant in the local and export fruit markets in Thailand. However, fruit rot, root rot, and stem rot diseases are caused by Phytophthora spp. such as P. palmivora, P. nicotianae, and Pythium cucurbitacearum, which cause huge damage to Durian cultivation through all stages of cultivation [51,52]. The economic losses due to crop loss and the prevention measures are calculated to be 20–25% of the total production [51]. The imports of oomycete fungicides have drastically increased from 10,988 tons (154 million USD) in 2014 to 21,004 tons (687 million USD) in 2018 [53,54]. The main fungicide groups for controlling the oomycete diseases in Durian are carboxylic acid amides (CAAs), phenylamides (PAs), and quinone outside inhibitors (QoIs). However, these fungicides are applied to the field extensively. In extreme cases, it is as high as 20 times within a year until the harvest [1]. In Thailand, the first metalaxyl resistance in Phytophthora spp. was observed with potato late blight caused by P. infestans [55]. Later in 2018, The Fungicide Resistance Action Committee (https://www.frac.info/ (accessed on 10 January 2019) reported the metalaxyl resistance to Phytophthora spp. in Thailand [1]. The recent study by Kongtragoul et al. [1] revealed the presence of metalaxyl resistant P. palmivora associated with ‘Monthong’ Durian cultivation in Thailand. The presence of resistant species demarcates the ineffectiveness of the desired fungicide. Kongtragoul and Viriyaekkul [56] found leaf fall disease in para-rubber caused by Phytophthora spp. in the same region where metalaxyl has been extensively (approximately 2-3 times/month or more often) used to control the Phytophthora diseases in Durian. Such extensive usage of fungicides results in the development of resistance to these fungicides [1]. The current disease’s onset was observed in the highlands of northern Thailand. Which covers 70% of Northern Thailand [57]. However, pesticides and agrochemical usage in the highlands caused adverse effects on the agroecosystem of the region. Further, through the Bio-Circular-Green Economy (BCG) model and the UN Sustainable Development Goals (SDGs), Thailand is heading toward pesticide-free agriculture [58]. Hence, it is important to explore alternative ways to reduce fungicide usage in Thailand.
The sample collection sites of the current study are highly affected by Phytophthora palmivora. The disease onset was observed on the well-established plants of the cultivation. Which caused huge losses to the farmers. For proper disease management and to research proper controlling measures, the correct identification of the pathogen is important [59]. Under the current study, the pathogenicity effects of the isolates have been proven through the satisfaction of Koch’s postulates. The identity of the pathogen and its taxonomic placement were identified through muti-gene phylogeny and morphology studies. The taxonomy and phylogeny identified the pathogen as Phytophthora palmivora.
In the current study, Vernonia amygdalina leaves were extracted into an EtOAc layer to obtain a wide range of compounds with different polarities. Most of the previous studies on disease control through biologically active compounds used nanoparticles as the compound delivery source [40]. However, microparticles and emulsions show prolonged effects with much greater stability and feasibility for production compared to extract loaded nanoparticles [40,60,61]. Although, the formation of microspheres through the electrospraying process depends on several variables. Variables such as the solution feeding rate, voltage, distance from the needle tip to the collector, type of collector, temperature, and humidity affect the particle size and morphology during the electrospraying process [62,63,64]. In this study, we studied the influence of electrospray process parameters, such as solution feeding rate, voltage, and needle tip to collector distance. The effect of the above variables was assessed using SEM. The results indicated that the higher voltage produced even-sized, smaller microspheres, while lower voltages produced larger and distorted microspheres. In our investigation, eight different schemes of electrospray-based microsphere production were investigated. According to Tapia-Hernández et al. [65], higher flow rates increase the solvent concentration in the droplets. Therefore, higher polymer concentrations increase the size of the resultant particle. This effect was well demonstrated in our results, where at 4 mL/h, no properly shaped particle was produced. The polymer aggregated or produced flaky particles. Further, the desired microspheres were obtained only at a rate of 2 mL/h. However, the electric potential also affects the uniformity of micro-particle size. Our experiment demonstrates that the EtOAc extract loaded microspheres drastically inhibited the growth of Phytophthora palmivora compared to the direct application of the EtOAc extract. The results proved that ED50 decreased to 61.10 µg/mL with the usage of EtOAc extract loaded micro-particles. However, more studies are needed to improve the current study for in vivo conditions. Further, our study leads the way to research plant extract based pesticide-free alternatives to support sustainable agriculture practices in Thailand.

Author Contributions

Conceptualization, R.C., P.T. and A.K.; methodology, J.C., K.Y., C.C., P.I., P.T., B.S., S.H., J.N. and A.K.; validation, C.T.-a., R.C., P.T. and A.K.; resources, R.C. and P.T.; writing—original draft preparation, J.C., A.K., P.T. and R.C.; writing—review and editing, A.K., P.T. and T.P.; supervision, R.C. and P.T.; project administration, R.C. and P.T.; funding acquisition, R.C. All authors have read and agreed to the published version of the manuscript.

Funding

The Project was partially funded by the Thailand Research Fund (SRI6020204).

Informed Consent Statement

Not applicable.

Data Availability Statement

Data is contained within the article.

Acknowledgments

The authors wish to thank the Department of Entomology and Plant Pathology, Faculty of Agriculture, Chiang Mai University, Chiang Mai, Thailand. This research is partially supported by the Post-Doctoral Fellowship 2022 for Reinventing Chiang Mai University. We would like to acknowledge Paweł Działak for his valuable comments on the project.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Kongtragoul, P.; Ishikawa, K.; Ishii, H. Metalaxyl Resistance of Phytophthora palmivora causing durian diseases in Thailand. Horticulturae 2021, 7, 375. [Google Scholar] [CrossRef]
  2. Durian: Situations of Important Agricultural Products and Trends. Available online: https://www.oae.go.th (accessed on 15 June 2021).
  3. Kwangtong, C. Farmers. Utilization of Durian Production Technology in Chanthaburi Province: A Case Study of Khlung District. Chanthaburi Province. Master’s Thesis, Kasetsart University, Bangkok, Thailand, 1998. [Google Scholar]
  4. Erwin, D.C.; Ribeiro, O.K. Phytophthora Diseases Worldwide; American Phytopathological Society (APS Press): St. Paul, MN, USA, 1996; ISBN 9780890542125. [Google Scholar]
  5. Ferrin, D.M.; Kabashima, J.N. In vitro insensitivity to metalaxyl of isolates of Phytophthora citricola and P. parasitica from ornamental host in southern California. Plant Dis. 1991, 75, 1041–1044. [Google Scholar] [CrossRef]
  6. Pongpisutta, R. Fruit Rot of Durian cv. Mon Thong Caused by Phytophthora palmivora (Butl.) Butl. and Its Control. Master’s Thesis, Kasetsart University, Bangkok, Thailand, 1992. [Google Scholar]
  7. Earnshaw, D.M.; Shattock, R.C. Sensitivity of Progeny of Phytophthora infestans to Fungicides. Asian J. Agric. Sci. 2012, 4, 213–224. [Google Scholar]
  8. Fontem, D.A.; Olanya, O.M.; Tsopmbeng, G.R.; Owona, M.A.P. Pathogenicity and metalaxyl sensitivity of Phytophthora infestans isolates obtained from garden huckleberry, potato, and tomato in Cameroon. J. Crop Prot. 2005, 24, 449–456. [Google Scholar] [CrossRef]
  9. Zhu, G.; Huang, F.; Feng, L.; Qin, B.; Yang, Y.; Hen, Y.; Lu, X. Sensitivities of Phytophthora infestans to metalaxyl, cymoxanil, and dimethomorph. Agric. Sci. China 2008, 7, 831–840. [Google Scholar] [CrossRef]
  10. Runno-Paurson, E.; Fry, W.E.; Remmel, T.; Mänd, M.; Myers, K.L. Phenotypic and genotypic characterisation of Estonian isolates of Phytophthora infestans in 2004–2007. J. Plant Pathol. 2010, 92, 375–384. [Google Scholar]
  11. Matuszak, J.M.; Fernandez-Elquezabel, J.; Gu, W.K.; Villarreal-Gonzalez, M.; Fry, W.E. Sensitivity of Phytophthora infestans populations to metalaxyl in Mexico: Distribution and dynamics. Plant Dis. 1994, 78, 911–916. [Google Scholar] [CrossRef]
  12. Lee, T.Y.; Mizubuti, E.; Fry, W.E. Genetics of metalaxyl resistance in Phytophthora infestans. Fungal Genet. Biol. 1999, 26, 118–130. [Google Scholar] [CrossRef]
  13. Hammi, A.; Msatef, Y.; Bennani, A.; Ismaili, A.E.L.; Serrhini, M.N. Mating type, metalaxyl resistance and aggressiveness of Phytophthora infestans (Mont.) de Bary in Morocco. J. Phytopathol. 2002, 150, 289–291. [Google Scholar] [CrossRef]
  14. Sobkowiak, S.; Śliwka, J.; Chmielarz, R.; Lebecka, R.; Zimnoch-Guzowska, E. Resistance to metalaxyl of Phytophthora infestans isolates occurring in Poland in 2006–2010. Phytopathologia 2016, 61, 29–35. [Google Scholar]
  15. Elansky, S.; Apryshko, V.; Milyutina, D.; Kozlovsky, B. Resistance of Russian strains of Phytophthora infestans to fungicides metalaxyl and dimethomorph. Moscow Univ. Biol. Sci. Bull. 2007, 62, 11–14. [Google Scholar] [CrossRef]
  16. Mukalazi, J.; Adipala, E.; Sengooba, T.; Hakiza, J.J.; Olanya, M.; Kidanemariam, H.M. Metalaxyl resistance, mating types, and pathogenicity of Phytophthora infestans in Uganda. Crop Prot. 2001, 20, 379–388. [Google Scholar] [CrossRef]
  17. Timmer, L.W.; Graham, J.H.; Zitko, S.E. Metalaxyl-resistant isolates of Phytophthora nicotianae: Occurrence, sensitivity, and competitive parasitic ability on citrus. Plant Dis. 1998, 82, 254–261. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  18. Parra, G.; Ristaino, J.B. Resistance to mefenoxam and metalaxyl among field isolates of Phytophthora capsici causing Phytophthora blight of bell pepper. Plant Dis. 2001, 85, 1069–1075. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  19. Taylor, R.J.; Salas, B.; Secor, G.A.; Rivera, V.; Gudmestad, N.C. Sensitivity of North American isolates of Phytophthora erythroseptica and Pythium ultimum to mefenoxam (metalaxyl). Plant Dis. 2002, 86, 797–802. [Google Scholar] [CrossRef] [Green Version]
  20. Peter, R.D.; Sturz, A.V.; Matheson, B.G.; Arsenault, W.J.; Malone, A. Metalaxyl sensitivity of isolates of Phytophthora erythroseptica in Prince Edward Island. Plant Pathol. 2001, 50, 302–309. [Google Scholar] [CrossRef]
  21. Kanchanaroek, Y.; Aslam, U. Policy schemes for the transition to sustainable agriculture-Farmer preferences and spatial heterogeneity in northern Thailand. Land Use Policy 2018, 78, 227–235. [Google Scholar] [CrossRef]
  22. Mahmood, I.; Imadi, S.R.; Shazadi, K.; Gul, A.; Hakeem, K.R. Effects of pesticides on environment. In Plant, Soil and Microbes: Volume 1: Implications in Crop Science; Hakeem, K., Akhtar, M., Abdullah, S., Eds.; Springer: Cham, Switzerland, 2016; pp. 253–269. ISBN 978-3-319-27455-3. [Google Scholar]
  23. Compant, S.; Duffy, B.; Nowak, J.; Clement, C.; Barka, E.A. Use of plant growth promoting bacteria for biocontrol of plant diseases: Principles, mechanisms of action, and future prospects. Appl. Environ. Microbiol. 2005, 71, 4951–4959. [Google Scholar] [CrossRef] [Green Version]
  24. Dowling, D.N.; Ogara, F. Metabolites of Pseudomonas involved in the biocontrol of plant disease. Trends Biotechnol. 1994, 12, 133–141. [Google Scholar] [CrossRef]
  25. Banerjee, S.; Mitra, A. Changing landscape of herbal medicine: Technology attributing renaissance. Int. J. Pharm. Pharm. Sci. 2012, 4, 47–52. [Google Scholar]
  26. Zhao, X.; Cui, H.; Wang, Y.; Sun, C.; Cui, B.; Zeng, Z. Development strategies and prospects of nano-based smart pesticide formulation. J. Agric. Food Chem. 2017, 66, 6504–6512. [Google Scholar] [CrossRef] [PubMed]
  27. Mora-Huertas, C.E.; Fessi, H.; Elaissari, A. Polymer-based nanocapsules for drug delivery. Int. J. Pharm. 2010, 385, 113–142. [Google Scholar] [CrossRef] [PubMed]
  28. Huang, S.; Chang, W. Advantages of nanotechnology-based Chinese herb drugs on biological activities. Curr. Drug Metab. 2009, 10, 905–913. [Google Scholar] [CrossRef] [PubMed]
  29. Ajazuddin; Saraf, S. Applications of novel drug delivery system for herbal formulations. Fitoterapia 2010, 81, 680–689. [Google Scholar] [CrossRef]
  30. Devi, V.K.; Jain, N.; Valli, K.S. Importance of novel drug delivery systems in herbal medicines. Pharmacogn. Rev. 2010, 4, 27–31. [Google Scholar] [CrossRef] [Green Version]
  31. Yen, F.L.; Wu, T.H.; Tzeng, C.W.; Lin, L.T.; Lin, C.C. Curcumin Nanoparticles improve the physicochemical properties of curcumin and effectively enhance Its antioxidant and antihepatoma activities. J. Agric. Food Chem. 2010, 58, 7376–7382. [Google Scholar] [CrossRef]
  32. Dar, J.; Soytong, K. Construction and characterization of copolymer nanomaterials loaded with bioactive compounds from Chaetomium species. J. Agric. Technol. 2014, 10, 823–831. [Google Scholar]
  33. Song, J.J.; Soytong, K.; Kanokmedhakul, S.; Kanokmedhakul, K. Nano-particles from Chaetomium lucknowense to inhibit rice blast pathogen causded by Pyricularia oryzae in pot experiment. Int. J. Agric. Technol. 2018, 14, 1961–1968. [Google Scholar]
  34. Malik, S.A.; Ng, W.H.; Bowen, J.; Tang, J.; Gomez, A.; Kenyon, A.J.; Day, R.M. Electrospray synthesis and properties of hierarchically structured PLGA TIPS microspheres for use as controlled release technologies. J. Colloid Interface Sci. 2016, 467, 220–229. [Google Scholar] [CrossRef] [Green Version]
  35. Liu, H.; Du, K.; Li, D.; Du, Y.; Xi, J.; Xu, Y.; Shen, Y.; Jiang, T.; Webster, T.J. A high bioavailability and sustained-release nano-delivery system for nintedanib based on electrospray technology. Int. J. Nanomed. 2018, 13, 8379–8393. [Google Scholar] [CrossRef] [Green Version]
  36. Raghavan, P.; Lim, D.-H.; Ahn, J.-H.; Nah, C.; Sherrington, D.C.; Ryu, H.-S.; Ahn, H.-J. Electrospun polymer nanofibers: The booming cutting edge technology. React. Funct. Polym. 2012, 72, 915–930. [Google Scholar] [CrossRef] [Green Version]
  37. Santos, J.; Silva, G.S.; Velho, M.C.; Beck, R.C.R. Eudragit®: A versatile family of polymers for hot melt Extrusion and 3D Printing Processes in Pharmaceutics. Pharmaceutics 2010, 13, 1424. [Google Scholar] [CrossRef] [PubMed]
  38. Alara, O.R.; Abdurahmana, N.H.; Mudalipa, S.K.A.; Olalerea, O.A. Phytochemical and pharmacological properties of Vernonia amygdalina: A review. J. Chem. Eng. Ind. Biotech. 2017, 2, 96. [Google Scholar] [CrossRef]
  39. Kadiri, O.; Olawoye, B. Vernonia amygdalina: An underutilized vegetable with nutraceutical Potentials—A Review. Turk. J. Agric. Food Sci. Technol. 2016, 4, 763–768. [Google Scholar] [CrossRef] [Green Version]
  40. Intaparn, P. Controlling Pythium Root Rot by Nano-Particle Extracts from Cordyceps and Herbs. Ph.D. Thesis, Chiang Mai University, Chiang Mai, Thailand, 2020. [Google Scholar]
  41. Yusoff, S.F.; Haron, F.F.; Tengku Muda Mohamed, M.; Asib, N.; Sakimin, S.Z.; Abu Kassim, F.; Ismail, S.I. Antifungal Activity and Phytochemical Screening of Vernonia amygdalina Extract against Botrytis cinerea Causing Gray mold disease on tomato fruits. Biology 2020, 9, 286. [Google Scholar] [CrossRef] [PubMed]
  42. Yusoff, S.F.; Haron, F.F.; Asib, N.; Mohamed, M.T.M.; Ismail, S.I. Development of Vernonia amygdalina leaf extract emulsion formulations in controlling gray mold disease on tomato (Lycopersicon esculentum Mill.). Agronomy 2021, 11, 373. [Google Scholar] [CrossRef]
  43. Barrero, A.F.; Oltra, J.E.; Álvarez, M.; Raslan, D.S.; Saúde, D.A.; Akssira, M. New sources and antifungal activity of sesquiterpene lactones. Fitoterapia 2000, 71, 60–64. [Google Scholar] [CrossRef]
  44. Wedge, D.E.; Galindo, J.C.G.; Macõâ, F.A. Fungicidal activity of natural and synthetic sesquiterpene lactone analogs. Phytochemistry 2000, 53, 747–757. [Google Scholar] [CrossRef]
  45. Erasto, P.; Grierson, D.S.; Afolayan, A.J. Bioactive sesquiterpene lactones from the leaves of Vernonia amygdalina. J. Ethnopharmacol. 2006, 106, 117–120. [Google Scholar] [CrossRef]
  46. White, T.J.; Bruns, T.; Lee, S.; Taylor, J. Amplication and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In PCR Protocols: A Guide to Methods and Applications; Innis, M.A., Gelfand, D.H., Sninsky, J.J., White, T.J., Eds.; Academic Press: New York, NY, USA, 1990; pp. 315–322. [Google Scholar]
  47. Robideau, G.P.; Cock, A.M.; Coffey, M.D.; Voglmayr, H.; Robideau, G.P.; Cock, A.W. DNA barcoding of oomycetes with cytochrome c oxidase subunit I and internal transcribed spacer. Mol. Ecol. Resour. 2011, 11, 1002–1011. [Google Scholar] [CrossRef]
  48. Wongwan, T.; Haituk, S.; Senwanna, C.; To-anun, C.; Intaparn, P.; Cheewangkoon, R. New host record of Phytophthora palmivora causing black rot on Rhynchostylis gigantea in Thailand. Chiang Mai J. Sci. 2021, 48, 942–951. [Google Scholar]
  49. Pharmacopeia. Optical Microscopy. Available online: http://www.pharmacopeia.cn/v29240/usp29nf24s0_c776.html (accessed on 14 June 2021).
  50. Suzui, T.; Kueprakone, U.; Kamphangridthrong, T. Phytophthora spp. isolated from some economic plants in Thailand. Tech. Bull. TRAC 1979, 12, 32–41. [Google Scholar]
  51. Drenth, A.; Guest, D.I. Diversity and Management of Phytophthora in Southeast Asia; ACIAR Monograph 114; Australian Centre for International Agricultural Research: Canberra, Australia, 2004. [Google Scholar]
  52. Suksiri, S.; Laipasu, P.; Soytong, K.; Poeaim, S. Isolation and identification of Phytophthora sp. And Pythium sp. from durian orchard in Chumphon province, Thailand. Int. J. Agric. Technol. 2018, 14, 389–402. [Google Scholar]
  53. Office of Agricultural Economics, Department of Agriculture, Thailand. Quantity and Value of Imports of agricultural Hazardous Substances. Resource Document Office of Agricultural Economics, Department of Agriculture, Thailand. 2018. Available online: http://oldweb.oae.go.th/economicdata/pesticides.html (accessed on 28 September 2019).
  54. Department of Agriculture Thailand. Durian Production Technology for Quality. Resource Document. 2018. Available online: http://www.doa.go.th/hrc/chantaburi/images/files/tecno_durian56.pdf (accessed on 10 January 2019).
  55. Chiampiriyakul, P.; Sopee, J.; Mekmok, T. Evaluation of metalaxyl sensitivity among Phytophthora infestans by poisoned food technique using corn agar. In Proceedings of the 49th Kasetsart University Annual Conference, Bangkok, Thailand, 1–4 February 2011; Volume 1, pp. 480–487. [Google Scholar]
  56. Kongtragoul, P.; Viriyaekkul, O. Sensitivity detection of Phytophthora spp. causing para-rubber leaf fall disease to same systemic fungicides. In Proceedings of the 2nd International Symposium on Agricultural Technology Global Agriculture Trends for Sustainability, Pattaya, Thailand, 1–3 July 2015; pp. 149–152. [Google Scholar]
  57. Black, R.; Jonglaekha, N.; Thanormthin, V. Problems concerning pesticide use in highland agriculture, Northern Thailand. In Management of Pests and Pesticides; CRC Press: Boca Raton, FL, USA, 2019; pp. 28–37. [Google Scholar]
  58. National Science and Technology Development Agency (NSTDA). Bio-Circular-Green Economy (BCG). Available online: https://www.bcg.in.th/eng/background/ (accessed on 14 June 2023).
  59. Cheewangkoon, R.; Kodsueb, R.; Monkhung, S.; Karunarathna, K.V.A.; Wongwan, T.; Withee, P.; Tamakeaw, N. Phytopathogenic fungi identification in Thailand: Current trends and future perspectives. Chiang Mai J. Sci. 2021, 48, 726–759. [Google Scholar]
  60. Steipel, R.T.; Gallovic, M.D.; Batty, C.J.; Bachelder, E.M.; Ainslie, K.M. Electrospray for generation of drug delivery and vaccine particles applied in vitro and in vivo. Mater. Sci. Eng. C 2019, 105, 110070. [Google Scholar] [CrossRef]
  61. Ghasemiyeh, P.; Mohammadi-Samani, S. Potential of nanoparticles as permeation enhancers and targeted delivery options for skin: Advantages and disadvantages. Drug Des. Dev. Ther. 2020, 14, 3271–3289. [Google Scholar] [CrossRef]
  62. Yurteri, C.U.; Hartman, R.P.; Marijnissen, J.C. Producing pharmaceutical particles via electrospraying with an emphasis on nano and nano structured particles—A review. KONA Powder Part. J. 2010, 28, 91–115. [Google Scholar] [CrossRef] [Green Version]
  63. Almería, B.; Deng, W.; Fahmy, T.M.; Gomez, A. Controlling the morphology of electrospray-generated PLGA microparticles for drug delivery. J. Colloid Interface Sci. 2010, 343, 125–133. [Google Scholar] [CrossRef] [PubMed]
  64. Soares, R.M.D.; Siqueira, N.M.; Prabhakaram, M.P.; Ramakrishna, S. Electrospinning and electrospray of bio-based and natu-ral polymers for biomaterials development. Mater. Sci. Eng. C Mater. Biol. Appl. 2018, 92, 969–982. [Google Scholar] [CrossRef]
  65. Tapia-Hernández, J.A.; Torres-Chávez, P.I.; Ramírez-Wong, B.; Rascón-Chu, A.; Plascencia-Jatomea, M.; Barreras-Urbina, C.G.; Rangel-Vazquez, N.A.; Rodríguez-Félix, F. Micro- and nanoparticles by electrospray: Advances and applications in Foods. J. Agric. Food Chem. 2015, 63, 4699–4707. [Google Scholar] [CrossRef]
Sustainability 15 10842 g001 550
Figure 1. Diseases of Durio zibethinus observed in the field: (A) dieback; (B–D) partial necrosis of the trunk (the treated areas are visible in red); (E) bark pieces moistened with exudates; (F) affected roots; (G) root rot exposing the lighter inner tissues.
Figure 1. Diseases of Durio zibethinus observed in the field: (A) dieback; (B–D) partial necrosis of the trunk (the treated areas are visible in red); (E) bark pieces moistened with exudates; (F) affected roots; (G) root rot exposing the lighter inner tissues.
Sustainability 15 10842 g001
Sustainability 15 10842 g002 550
Figure 2. Phytophthora palmivora (Ph 410): (A) culture on PDA; (B) sporangia; (C) zoospore; (D) oogonium; (E) chlamydospore; and (F) mycelia. Scale bars: (B–E) = 20 µm, (F) = 100 µm.
Figure 2. Phytophthora palmivora (Ph 410): (A) culture on PDA; (B) sporangia; (C) zoospore; (D) oogonium; (E) chlamydospore; and (F) mycelia. Scale bars: (B–E) = 20 µm, (F) = 100 µm.
Sustainability 15 10842 g002
Sustainability 15 10842 g003 550
Figure 3. The disease severity of some Phytophthora strains.
Figure 3. The disease severity of some Phytophthora strains.
Sustainability 15 10842 g003
Sustainability 15 10842 g004 550
Figure 4. Maximum likelihood (RAxML) based on analysis of a combined dataset of ITS and Cox1 sequence data. The tree is rooted to Phytopythium chamaehyphon and Phytopythium sindhum. The newly generated sequences are in red. The ex-type strains are in bold.
Figure 4. Maximum likelihood (RAxML) based on analysis of a combined dataset of ITS and Cox1 sequence data. The tree is rooted to Phytopythium chamaehyphon and Phytopythium sindhum. The newly generated sequences are in red. The ex-type strains are in bold.
Sustainability 15 10842 g004
Sustainability 15 10842 g005 550
Figure 5. Phytophthora palmivora (Ph 410) growth assay in (A) crude extract poison media and (B) crude extract loaded microspheres poison media.
Figure 5. Phytophthora palmivora (Ph 410) growth assay in (A) crude extract poison media and (B) crude extract loaded microspheres poison media.
Sustainability 15 10842 g005
Sustainability 15 10842 g006 550
Figure 6. Electrospray particle morphology under SEM at 2000× magnification: (A) flow rate of 2 mL/h, voltage of 15 kV, and distance of 12 cm; (B) flow rate of 2 mL/h, voltage of 15 kV, and distance of 15 cm; (C) flow rate of 2 mL/h, voltage of 20 kV, and distance of 12 cm; (D) flow rate of 2 mL/h, voltage of 20 kV, and distance of 15 cm. Scale bars: (A–D) = 10 µm.
Figure 6. Electrospray particle morphology under SEM at 2000× magnification: (A) flow rate of 2 mL/h, voltage of 15 kV, and distance of 12 cm; (B) flow rate of 2 mL/h, voltage of 15 kV, and distance of 15 cm; (C) flow rate of 2 mL/h, voltage of 20 kV, and distance of 12 cm; (D) flow rate of 2 mL/h, voltage of 20 kV, and distance of 15 cm. Scale bars: (A–D) = 10 µm.
Sustainability 15 10842 g006
Sustainability 15 10842 g007 550
Figure 7. SEM images of electrospray particles at 2000 times magnification (A) 0.5% w/v, (B) 1% w/v, (C) 3% w/v, and (D) 5% w/v. Scale bars: (A–D) = 10 µm.
Figure 7. SEM images of electrospray particles at 2000 times magnification (A) 0.5% w/v, (B) 1% w/v, (C) 3% w/v, and (D) 5% w/v. Scale bars: (A–D) = 10 µm.
Sustainability 15 10842 g007
Sustainability 15 10842 g008 550
Figure 8. Histogram of 3% w/v Eudragit® E particle size.
Figure 8. Histogram of 3% w/v Eudragit® E particle size.
Sustainability 15 10842 g008
Table
Table 1. Isolates of Phytophthora spp. from various sites and pathogenic effects in Durian leaves.
Table 1. Isolates of Phytophthora spp. from various sites and pathogenic effects in Durian leaves.
IsolatesLocationSamplePathogenicity * (cm)
Ph201Chiang Daosoil0
Ph202Chiang Daosoil0
Ph203Chiang Daosoil0.2
Ph204Chiang Daosoil0
Ph205Chiang Daosoil0
Ph206Chiang Daosoil0.2
Ph207Chiang Daosoil0
Ph408Chiang Daoroot0
Ph409Chiang Daoroot0.3
Ph410Mae Hiaroot1.8
Ph211Mae Hiasoil0.2
Ph212Mae Hiasoil0
Ph213Mae Hiasoil0
Ph214Mae Hiasoil0
Ph215Mae Hiasoil0.3
Ph216Mae Hiasoil0
Ph217Mae Hiasoil0
Ph418Mae Hiaroot0
Ph419Mae Hiaroot0.4
Ph420Mae Hiaroot0.5
Ph421Mae Hiaroot0
Ph422Mae Hiaroot0.2
Ph423Mae Hiaroot0
Ph424Mae Hiaroot0
Ph225Mae Aisoil0.4
Ph226Mae Aisoil0
Ph427Mae Airoot0
Ph428Mae Airoot0
Ph429Mae Airoot0.3
Ph430Mae Airoot0
* The diameter of the lesion: 0 = nonpathogenic, 0.1–0.5 = virulent (level 1), 0.6–1.0 = virulent (level 2), 1.1–1.5 = virulent (level 3), and >1.6 = virulent (level 4).
Table
Table 2. Percentage of suppression and Effective Dose 50 in inhibiting growth of P. palmivora (Ph 410).
Table 2. Percentage of suppression and Effective Dose 50 in inhibiting growth of P. palmivora (Ph 410).
ExtractConcentrations (µg/mL)Inhibiting Growth of Phytophthora sp. (Ph 410)
PIRG (%)ED50 (µg/mL)
Crude extract0-374.87
1018.44
5031.56
10047.78
50072.22
100085.33
Particles containing crude extract0-61.10
18.67
54
105.11
5044.22
10089.56
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Chainanta, J.; Yakajay, K.; Chinda, C.; Intaparn, P.; To-anun, C.; Tipduangta, P.; Sirithunyalug, B.; Haituk, S.; Nguanhom, J.; Pusadee, T.; et al. Vernonia amygdalina Extract Loaded Microspheres for Controlling Phytophthora palmivora. Sustainability 2023, 15, 10842. https://doi.org/10.3390/su151410842

AMA Style

Chainanta J, Yakajay K, Chinda C, Intaparn P, To-anun C, Tipduangta P, Sirithunyalug B, Haituk S, Nguanhom J, Pusadee T, et al. Vernonia amygdalina Extract Loaded Microspheres for Controlling Phytophthora palmivora. Sustainability. 2023; 15(14):10842. https://doi.org/10.3390/su151410842

Chicago/Turabian Style

Chainanta, Jiratchaya, Kittiporn Yakajay, Chanakarn Chinda, Phikul Intaparn, Chaiwat To-anun, Pratchaya Tipduangta, Busaban Sirithunyalug, Sukanya Haituk, Jeerapa Nguanhom, Tonapha Pusadee, and et al. 2023. "Vernonia amygdalina Extract Loaded Microspheres for Controlling Phytophthora palmivora" Sustainability 15, no. 14: 10842. https://doi.org/10.3390/su151410842

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop