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Variability of Lecanicillium spp. Mycoparasite of Coffee Leaf Rust Pathogen (Hemileia vastatrix) in Indonesia



Roosmarrani Setiawati, Ani Widiastuti, Arif Wibowo and Achmadi Priyatmojo
 
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ABSTRACT

Background and Objective: Coffee leaf rust disease caused by Hemileia vastatrix resulted in high yield loss and difficult to control. Several chemical fungicides have been used to control this disease. However, the effectiveness of chemical control is low, so it is necessary to find other methods such as biological control. Lecanicillium spp. is well-known as mycoparasite on H. vastatrix uredospores but the study in Indonesia is still limited. This study aimed to collect and investigated the genetic variability of Lecanicillium spp. at various coffee plantations in Indonesia. Materials and Methods: Samples of Lecanicillium spp. were collected from 20 districts in 7 provinces throughout Indonesia. Morphology of colony and conidia were identified by visual examination and by viewed under the light microscope. Genetic variability was conducted using Rep-PCR and clustered with UPGMA. Results: Morphological observation in this study revealed all isolates collected from uredospores of H. vastatrix were similar with Lecanicillium spp. Genetic variability analysis clustered the 80 isolates into eight clusters with their specific characters. Conclusion: Morphological identification in this study showed that 80 isolates of mycoparasite on H. vastatrix belong to Lecanicillium spp. Further study using the molecular technique is needed to identity the species of Lecanicillium.

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Roosmarrani Setiawati, Ani Widiastuti, Arif Wibowo and Achmadi Priyatmojo, 2021. Variability of Lecanicillium spp. Mycoparasite of Coffee Leaf Rust Pathogen (Hemileia vastatrix) in Indonesia. Pakistan Journal of Biological Sciences, 24: 588-598.

DOI: 10.3923/pjbs.2021.588.598

URL: https://scialert.net/abstract/?doi=pjbs.2021.588.598
 
Copyright: © 2021. This is an open access article distributed under the terms of the creative commons attribution License, which permits unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited.

INTRODUCTION

Coffee (Coffea spp.) is one of the world’s main plantation commodities on which many countries rely as a source of foreign exchange1. Indonesia is the fourth largest coffee producer in the world with a planted area of 1.2 million ha and total production of 639,305 t in 20172.

Coffee productivity in Indonesia is still relatively low when compared to other coffee producing countries. The main obstacles in the development of coffee in Indonesia are coffee leaf rust disease caused by Hemileia vastatrix (Berkeley and Broome). The loss caused by this disease may reach 70% in susceptible varieties3.

Conventional control over this disease is carried out with the application of fungicides4. Several types of fungicidal active ingredients recommended by the Pesticide Commission in Indonesia are cyproconazole, hexacazanole, triadimefon, benomyl, copper oxychloride, mancozeb, copper hydroxide, copper oxide, difenoconazole and propiconazole. This application only reduces the rate of plant damage on coffee leaf rust disease by about 20%5. The use of the systemic fungicide triadimefon should be limited because it can affect plant physiology, especially photosynthetic6.

Unfortunately, this application method not only causing pathogen resistance to fungicides but also causing phytotoxicity, resulting in the emergence of a new physiological race of H. vastatrix and residue in the surrounding environment4,7. Therefore, it is necessary to use alternative disease control in coffee using biological control agents to control coffee leaf rust disease8.

Lecanicillium spp. (formerly Verticillium spp.) is well-known as entomopathogens that have been commercially developed as a biopesticide. This fungus produces toxic secondary metabolites, namely bassianolidae and dipicolinic acid, which are insecticidal9-11. However, recently is has been discovered that this entomopathogen is also known as mycoparasite in some pathogenic fungi such as Sphaerotheca fuliginea, S. macularis f. sp. fragariae, Penicillium digitatum and Phytium ultimum11-15. Several species of the Lecanicillium also have been reported to parasitize H. vastatrix uredospores4,16-21.

Lecanicillium spp. was able to control H. vastatrix in the laboratory and in the greenhouse18. In vitro, this reduced uredospores of H. vastatrix up to 59.3% and the rate of crop damage due to H. vastatrix decreased by 68.2%. Gomez-De La Cruz et al.22 also stated that Lecanicillium spp. showed mycoparasitism in coffee leaf rust uredospores, 120 hrs after inoculation with an average of 68%. The potential of

Lecanicillium spp. as a biological control agent of H. vastatrix needs to be further investigated as an environmentally friendly control effort.

The purpose of this study was to collect and find out the genetic variability of Lecanicillium spp. associated with H. vastatrix isolated from a various coffee plantations in Indonesia.

MATERIALS AND METHODS

Study area: The study was carried out at the Laboratory of Plant Disease, Department of Crop Protection, Faculty of Agriculture, Universitas Gadjah Mada, Special Region of Yogyakarta, Indonesia since August, 2019-October, 2020.

Sample collection: Samples of Lecanicillium spp. were collected by purposive sampling at coffee plantations in seven provinces in Indonesia including West Java, Central Java, Special Region of Yogyakarta, East Java, Bali, West Nusa Tenggara (NTB) and East Nusa Tenggara (NTT) (Fig. 1). Coffee leaf samples showing lesions and containing uredospores of H. vastatrix with colonies of Lecanicillium spp. on top were plucked from the coffee plant. The leaves are put in a paper bag and store in a cooler box before taken to the laboratory for isolation.

Isolation of Lecanicillium spp.: Isolation of Lecanicillium spp. was done aseptically by taking white mycelium using a needle preparation and growing it into a petri dish containing 2% Water Agar (WA) medium. After incubation for 3-5 days, purification was carried out by inoculating the growing mycelium on the WA into a petri dish containing Potato Dextrose Agar-L (PDA added 1-2 drops of 25% lactic acid). Incubation was carried out at room temperature (25-27°C) for 7 days.

Purification of Lecanicillium spp. with single-spore isolation: Lecanicillium spp. single spore isolation was carried out by cutting using 0.5 cm cork borer of 7 days culture on PDA and adding with 1 mL sterile distilled water in 1.5 mL tube. The suspension was homogenized by Thermolyne Mixer vortex (Haverhill, MA, USA) for about 2 min. The suspension obtained was taken using a loop needle and scratched on the new PDA medium. The streaks were incubated for approximately 12-24 hrs at 25-27°C. The germinated fungal spore then took by examination through a light microscope using a sterile needle and transferred into new PDA plates.

Image for - Variability of Lecanicillium spp. Mycoparasite of Coffee Leaf Rust Pathogen (Hemileia vastatrix) in Indonesia
Fig. 1:Sampling sites Lecanicillium spp. associated with Hemileia vastatrix
  Map resource: RBI Map 2010

Pure cultures were identified by visual examination (macroscopic) and viewed under the CX31 microscope (Olympus Co., Tokyo, Japan) (microscopic).

Morphological analysis: Morphological characteristics of the isolates were observed based on culture growth on PDA which incubated at 25-27°C for 21 days18,23. The observation of culture morphology including colony color, colony texture and colony diameter visually. Fungal colony colors were observed from the lower side of the culture plate and compare with the Munsell Soil Color Charts after three weeks of incubation. Microscopic features were observed by slide cultures on PDA, included conidiophores, crystal and conidia size. Fifty conidia were observed for shape and size measures. The examination was done using CX 31 microscope (Olympus Co., Tokyo, Japan), an image captured by DP 22 microscope camera (Olympus Co., Tokyo, Japan) and viewer cellSens imaging software (Olympus Co., Tokyo, Japan).

DNA extraction: Mycelia of 80 sample isolate for DNA extraction were grown on Potato Dextrose Broth (PDB: potato 200 g: Dextrose 20 g: Water to a final volume of 1,000 mL) in Erlenmeyer and shaken for 7 days at 25-27°C. Each DNA was extracted using the Cetyl Trimethyl Ammonium Bromide (CTAB) according to Xia et al.24 with modification. 0.5 g of mycelia were broken done by grinding by a mortal in a porcelain with the addition of 700 μL of 2% CTAB (2% PVP, 100 mM Tris-HCl pH 8, 25 mM EDTA, 2 M NaCl and 200 mM beta-mercaptoethanol/BME CTAB) solution and a little quartz sand. The mixture was then put into an Eppendorf tube 1.5 mL, then heated in a water bath at 60°C, for 30 min (shaken every 10 min) and centrifuged at 5000 rpm for 5 min. The supernatant was recovered into a 1.5 mL microtube, then added with CIAA (chloroform: isoamyl alcohol 24:1,v/v)) added until the tube was full, followed by centrifugation for 10 min at 12,000 rpm. The top supernatant fraction was recovered into a new microtube, to which, absolute ethanol was added and stored overnight at -20°C. Subsequently, the fraction was centrifuged for 10 min at 12,000 rpm. The resultant supernatant was removed and the pellet was washed in 70% ethanol and further centrifuged for 10 min at 12,000 rpm. The pellet was air dried for 2-3 hrs and re-suspended in 20-50 μL of pure water.

Table 1:Primer sets for rep-PCR used in this study
Image for - Variability of Lecanicillium spp. Mycoparasite of Coffee Leaf Rust Pathogen (Hemileia vastatrix) in Indonesia

PCR amplification: The PCR was carried out in a total volume 25 μL, comprised of 12.5 μL DNA Taq polymerase (My Taq HS Red Mix, Bioline, London, United Kingdom), 1.5 μL each primer (100 μM) 1 μL DNA template and sterile water in a total volume of 25 μL. Amplification was conducted using T100 Thermal Cycler (Biorad, California, United States). The rep-PCR using BOX A1R and ERIC with the PCR program conditions in Table 1. Reactions were performed in BioRad T100'tm Thermal Cycler (Bio-Rad, Hercules, CA, USA). The PCR products were then visualized using 1.5% agarose gel (0.45 g of agarose (Molecular Biology Grade, Vivantis) in 30 mL of TBE 1X). Agarose gel was then electrophoresed at 100 V for 25 min using BioRad Mini ReadySub-Cell gt Horizontal Electrophoresis Cell (Bio-Rad, Hercules, CA, USA). Agarose gel was then stained with a solution of ethidium bromide for 10 min and washed with water. The result was then visualized under UV light. The DNA bands were then captured using a digital camera25.

Phylogenetic analysis: The band patterns were analyzed by summarizing them into table with notification of 0-1 (0 for no band, 1 for the present band). This was made using Microsoft Excel to place the band arrangement in columns and isolate numbers in rows to construct the dendrogram26. Dendrogram representing the genetic variability among Lecanicillium spp. were generated from the similarity matrices by applying unweighted pair-group arithmetic (UPGMA) mean methods in numerical taxonomy and multivariate analysis system (NTSYSpc version 2.10e).

RESULTS

Study area: Samples collection of Lecanicillium spp. was conducted at 20 districts in 7 provinces throughout Indonesia with various altitudes (Table 2). Altitude varied from 669-1620 m above sea level. Its host was H. vastatrix with varied varieties origin of coffee, namely Coffea arabica L., C. canephora Pierre and C. liberica var dewevrei.

Symptoms of rust on coffee leaves are shown in Fig. 2a, b. In general, a symptom of rust on coffee leaves occur on the lower leaf surface. The initial symptom is in the form of chlorotic spots and will continue with the formation of a light yellow necrotic lesion which gradually turns dark yellow like rust. These lesions form orange pustules, which are masses of H. vastatrix uredospores (Fig. 2a). Lecanicillium spp. is one of the mycoparasite associated with H. vastatrix caused coffee rust disease. This fungal infection is characterized by initial symptoms of white colonies on H. vastatrix uredospores that form on the surface of the coffee leaves. The occurrence of these parasites causes less infection of H. vastatrix (Fig. 2b).

Morphological feature: Morphological characteristics of Lecanicillium spp. were observed on PDA after 21 days of incubation. Colonies reached 32-68 mm diameter in 21 days at 25-27°C. Texture colony of isolates was mostly wholly (Fig. 3a) and cottony (Fig. 3b), dense colony with aerial mycelia. Only a few isolates were velvety (Fig. 3c). The color of the aerial view of the isolates was white but the color of the reverse view varied, such as pale yellow, yellow, white, light brown and dark brown. Conidiophores are single or in groups of 2-4 phialides (verticillate) (Fig. 3d, e). Conidia formed in heads at the apex of phialides, 1.7-2.2×2.7-4.4 μm. Conidia produced by Lecanicillium spp. isolates were cylindrical or ellipsoidal (Fig. 3f). Crystals present with octahedral in shape (Fig. 3g).

Phylogenetic analysis using rep-PCR: Phylogenetic analysis was grouping 80 Lecanicillium spp. isolates the polymorphic bands ranging between 100-5000 bp were observed. UPGMA analysis using BOX A1R and ERIC primers showed that there were 8 clusters of Lecanicillium spp. at a coefficient value of 0.80 (Fig. 4). The data of Table 3 showed cluster 1 consisting of 26 isolates, cluster 2 consisting of 17 isolates, cluster 3 consisting of 14 isolates, cluster 4 consisting of 9 isolates, cluster 5 consisting of 4 isolates, cluster 6 consisting of 3 isolates cluster 7 consisting of 5 isolates and cluster 5 which only consisted of 2 isolates, namely DIY 4 and NTB 3.

Table 2:List of Lecanicillium spp. isolates
Image for - Variability of Lecanicillium spp. Mycoparasite of Coffee Leaf Rust Pathogen (Hemileia vastatrix) in Indonesia
Image for - Variability of Lecanicillium spp. Mycoparasite of Coffee Leaf Rust Pathogen (Hemileia vastatrix) in Indonesia

Image for - Variability of Lecanicillium spp. Mycoparasite of Coffee Leaf Rust Pathogen (Hemileia vastatrix) in Indonesia
Fig. 2:
Rust symptom on coffee leaf caused by Hemileia vastatrix (a), The white colonies fungus, Lecanicillium spp. associated with Hemileia vastatrix (b)

Image for - Variability of Lecanicillium spp. Mycoparasite of Coffee Leaf Rust Pathogen (Hemileia vastatrix) in Indonesia
Fig. 3:Morphology of Lecanicillium spp.
 
Texture colony wholly (a); cottony (b); velvety (c); 21d on PDA; Conidiophores and phialides. (d-e); Conidia (f); Octahedral Crystal (g)

Table 3:List of Lecanicillium spp. isolates clusters based on UPGMA
Image for - Variability of Lecanicillium spp. Mycoparasite of Coffee Leaf Rust Pathogen (Hemileia vastatrix) in Indonesia
Sign* was represent to selected isolate

Image for - Variability of Lecanicillium spp. Mycoparasite of Coffee Leaf Rust Pathogen (Hemileia vastatrix) in Indonesia
Fig. 4: Dendrogram of Lecanicillium spp. isolates clusters based on UPGMA

Table 4:Colony and conidia are characteristic of each Lecanicillium spp. cluster
Image for - Variability of Lecanicillium spp. Mycoparasite of Coffee Leaf Rust Pathogen (Hemileia vastatrix) in Indonesia

Eight Lecanicillium spp. isolates were randomly selected as representatives from each cluster based on the phylogenetic tree to determine their genetic variation. The data of Table 4 showed that the 8 clusters had variation macroscopic and microscopic morphology. Cluster 1 was represented by JBR 6 isolate that had a white aerial view and reverse view colony. The texture colony was whole with a slow growth rate. Cluster 2 was represented by NTT 5 isolate that had cottony colony texture and a small size of conidia. Cluster 3 was represented by JTM 1 isolate that had light brown reverse view colony color, cottony with the medium growth rate. Cluster 4 was represented by JTM 10 isolate that had yellow reverse view colony color, wholly with a medium growth rate. Cluster 5 was represented by JTH 12 isolate that had a white aerial view and reverse view colony color, wholly with a medium growth rate. Cluster 6 was represented by JTH 15 isolate that had a big size of conidia and a fast growth rate. Cluster 7 was represented by DIY 5 isolate that had velvety colony texture and medium growth rate. Cluster 8 was represented by NTB 3 isolate that had a white aerial view and dark brown reverse view colony color. This cluster also had an ellipsoidal shape of conidia and a wholly texture colony. All isolates produced crystal with octahedral shape.

DISCUSSION

Recently, biological control using Lecanicillium spp. has been reported to manage some pathogenic fungi. Result of this study indicated that Lecanicillium spp. isolated from 7 provinces in Indonesia showed variations in macroscopic and microscopic morphology. Genus Lecanicillium formerly named Verticillium, can be identified by their morphological characteristics, despite it was very general. According to Zare et al.27 genus Verticillium is divided into 2 groups, namely Verticillium section Verticillium and Verticillium section Prostata. Most entomopathogen and fungicolous were included in Verticillium section Prostata, while plant pathogenic fungi were included in Verticillium section Verticillium. In 2001, Zare and Gams28 transferred a part of the species formerly classified in Verticillium sect. Prostrata into genus Lecanicillium and Simplicillium.

Results have indicated that Lecanicillium spp. is widespread. Those can be found on H. vastatrix in the coffee plantation centers throughout Indonesia regions at various altitudes.

Lecanicillium spp. also can be found associated with H. vastatrix on various varieties origin of coffee. These fungi formed white colonies on H. vastatrix uredospores. The occurrence of these parasites causes less infection of H. vastatrix (Fig. 2). According to Vandermeer et al.29, this hyperparasite is capable of reducing spore viability and disease severity. Therefore, Lecanicillium spp. has the potential to be developed as a biological control agent of H. vastatrix.

The morphological characteristic of this mycoparasite isolated from H. vastatrix uredospores in Indonesia was generally similar to those reported in literature30-33. This fungus is characterized by verticillate branching ofconidiophores. It arises in whorls on the upper portions of conidiophores. Conidia adhering in a globose slimy heads. All of them referred to the genus Lecanicillium.

Results based on UPGMA indicated that there are genetic variations among Lecanicillium spp. isolates. But this genetic variation is not related to the altitude of sample location or the distribution of the Lecanicillium spp. which tends to be random. Distribution of Lecanicillium spp. in clusters is thought to be influenced by environmental factors such as temperature, humidity, light and the interactions between them. Besides, the existence of the H. vastatrix physiological race is also thought to allow the formation of genetic variations of the mycoparasitic fungi. Recently, it was known as many as more than 49 races physiology of H. vastatrix34,35. This could be a further interesting research study.

Morphologically, the eight representatives of Lecanicillium spp. isolates are indistinguishable. An observation based on the morphology of fungi is less accurate because most of fungi cannot be distinguished morphologically. A molecular identification technique needs to be done to identify fungal species through phylogenetic analysis. However, it can be used as supporting data for characteristics of the fungus36. Therefore, further molecular analyses based on DNA sequences are required for identifying these isolates.

CONCLUSION

Morphological identification in this study showed that 80 isolates of mycoparasite on H. vastatrix belong to Lecanicillium spp. These fungi were widespread and found in 7 provinces of Indonesia. Based on Rep-PCR Lecanicillium spp. had genetic variation divided into 8 clusters. This study will be beneficial for development of eco-friendly disease management of coffee rust disease and will be continued for sequencing at the species level.

SIGNIFICANCE STATEMENT

This study discovers the potency of the Lecanicillium spp. obtained from 7 provinces in Indonesia as mycoparasite of Hemileia vastatrix, the causal pathogen of coffee leaf rust disease. Thus, this research provides basic information for the further research to uncover the areas of the variation of Lecanicillium spp.

ACKNOWLEDGMENT

This study has been funded by the Agency for the Extension and Development of Agricultural Human Resources (BPPSDMP) Ministry of Agriculture of the Republic of Indonesia (contract number 1645/KP.320/J/3/14). We thank to Mr. Saipul Abbas and Ms. Tiya Farisa Agustin for technical support.

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