INTRODUCTION
The imperfect fungus Verticillium dahliae (Kleb.) is a soil-borne phytopathogen. It is a highly destructive specie of the genus Verticillium that causes wilting in about 400 susceptible plant species in temperate and sub-tropical regions (Fradin & Thomma, 2006; Rehman, Su, Guo, Qi,
& Cheng, 2016; Tian & Kong, 2022). The fungus survives in soil and plant debris for a long time due to microsclerotia formation in the dead host plant (Kabir, Bhat, & Subbarao, 2004; Li et al., 2022). In the host plants, microsclerotia act as a source of inoculum for the infection of Verticillium. V. dahliae causes wilt disease in many plants, including vegetables, fruits, flowers, oilseed crops, fiber crops, and woody perennials (Table 1) (Berlanger
& Powelson, 2000). In addition, other economically important crops, including tobacco, are the host of V. dahliae infection.
Tobacco is an important cash crop in the Pakistani agriculture sector, which substantially contributes to the development of Pakistan’s economy. In Khyber Pakhtunkhwa, a major tobacco-producing province of Pakistan, a total of 32,500 hectares are planted. Khyber Pakhtunkhwa alone produces 77% of the total tobacco production in Pakistan (Ali et al., 2017). Although the tobacco yield in Pakistan is higher than in other tobacco- growing countries like Brazil, America, China, India, and Greece, the quality of tobacco is substandard.
It does not get a good price in the international market (Ali et al., 2017). Several factors stand responsible for this. It is inferred that the damage by V. dahliae affects the quality and quantity of tobacco.
Verticillium wilt disease is a major limiting factor of tobacco, severely impacting the plant agronomic performance and leading to economic losses. Two species of tobacco, Nicotiana tabacum L. and N.
Rustica L. are widely grown in Swabi, Mardan, ARTICLE INFO
Keywords:
Microsclerotia Morphology Tobacco
Verticillium dahliae Verticillium wilt Article History:
Received: October 27, 2021 Accepted: February 8, 2023
*) Corresponding author:
E-mail: [email protected]
ABSTRACT
Field and laboratory investigations of tobacco plants were carried out to analyze the distribution of Verticillium wilt caused by V. dahliae. V.
dahliae is isolated from field Z (Tarkhai, Yarhussain region of Swabi).
Species of Fusarium and Actinomycetes were detected in field B (Shaheedan, Yarhussain region of Swabi), while no V. dahliae was isolated from these fields. V. dahliae was identified based on its colony and conidia morphology. The colony was white, having oval spores and septate mycelium with phialides. The isolate also produced irregularly elongated dark brown to black color microsclerotia of various sizes. The spores count for V. dahliae isolate was 3.61 × 108 conidia/ml. The average colony diameter of V. dahliae after 9 days of incubation on PDA and CM media was 6.55 cm and 5.5 cm, respectively. The biology of Verticillium spp. in tobacco plants and soil in Swabi has not been studied. For the first time, this study reported the presence of V. dahliae in the tobacco fields of Swabi, KPK Pakistan. Detection and isolation of V. dahliae from field soil are necessary for studying the ecology and virulence and creating deterrent control strategies for Verticillium wilt diseases.
ISSN: 0126-0537Accredited First Grade by Ministry of Research, Technology and Higher Education of The Republic of Indonesia, Decree No: 30/E/KPT/2018
Cite this as: Ahmad, T., Akram, S., Fahad, S., Ilyas, M., Alam, M., Hussain, H., & Rehman, L. (2023). Assessment of tobacco fields for the prevalence of Verticillium dahliae in District Swabi, Pakistan. AGRIVITA Journal of Agricultural Science, 45(1), 69–78. http://doi.org/10.17503/agrivita.v45i1.3606
Assessment of Tobacco Fields for the Prevalence of Verticillium dahliae in District Swabi, Pakistan
Tasiq Ahmad1), Said Akram1), Shah Fahad1), Muhammad Ilyas2), Mukhtar Alam3), Hazrat Hussain1), and Latifur Rehman1*)
1) Department of Biotechnology, University of Swabi, Pakistan
2) Department of Botany, Kohsar University Murree, Pakistan
3) Department of Agriculture, University of Swabi, Pakistan
Peshawar, Swat, Buner, Charsadda, and Hazara districts (Syed, Khan, Khan, & Badshah, 2005).
V. alboatrum and V. dahliae were observed for the first time in hilly areas of Pakistan. Disease incidence of V. dahliae in Rawalpindi and Islamabad was detected in farmer’s potato fields (Ashraf, Rauf, Abbas, & Rehman, 2012). Environmental conditions in Swabi are suitable for the development of this disease. It is probable that Verticillium wilt is present and spread in the tobacco field of district Swabi.
The disease symptoms this study noted in different tobacco fields of Swabi were similar to those reported previously (Thomson & Mcleod, 1959;
Zhang, Dai, Klosterman, Subbarao, & Chen, 2022).
The first symptom was the wilting of one or more leaves, particularly in hot weather. It was followed by the interveinal area turning a bright orange color.
With the progression of the disease, the tissue dies and turns brown, leaving an orange border between the living and dead tissues. Symptoms-based diagnostics for the fungus are not confirmatory;
the fungal structure is not visible on the diseased specimen. Confirmation of the infection requires the use of laboratory tests.
The biology of Verticillium spp. in tobacco plants and soil in Swabi has not been studied.
Because tobacco is grown on very expensive land, with a high investment in equipment, it tends to be the main cash crop almost everywhere it is produced. Due to Verticillium wilt, the quality and quantity of N. tabacum are badly affected, resulting in extensive economic loss. Verticillium wilt appeared as a major disease of tobacco in February 1994 in New Zealand. The disease was so severe in some of these areas that economic tobacco production was no longer possible (Thomson & Mcleod, 1959). Latorre, Lolas, & Marholz (1989) conducted experiments to confirm the cause and importance of the outbreak in Burley tobacco fields in Chile. They estimated the effect of Verticillium wilt on yield in 11 commercial plots of tobacco cultivar Burley-49. The
study’s results confirmed that the outbreak of wilting and yellowing of tobacco plants was caused by V.
dahliae. Significant disease incidence and yield loss were estimated as high as 25.6% and 34.6%
simultaneously which resulted in 1440 kg/ha less yield than other symptomless plants.
Considering tobacco’s economic importance, the present study was designed to evaluate tobacco fields in different districts of Swabi for the isolation, detection, and morphological characterization of V. dahliae. The study will help improve tobacco production through farmers’ awareness to take necessary control measures. Similarly, this study will inform the scientific community to consider the disease’s contagious nature.
MATERIALS AND METHODS Study Site and Location
The present study was conducted in Swabi, Khyber Pakhtunkhwa, Pakistan, from 2020 to 2021. District Swabi is favorable for producing Desi and Flue Cured Virginia tobacco. There are many tobacco growers in this district. The study was based on soil sample collection from tobacco fields. Four fields in Yarhussain Zone II, previously noted as suspected for Verticillium wilt, were selected for the present study. Samples were collected from these four different regions of the selected area, Tarkha (N34º 06’16.6824”, E 72º14’04.2612”), Saheedan (N 34º 10’47.5644”, E 72º18’23.4252”), Jalaroona (N 34º 04’56.1756”, E 72º14’10.5288”), Jagannat (N 34º 05’47.6763”, E 72º 18’31.9572”). Random soil samples were collected from April-May from these fields. The method previously reported by Goddard (1913) was slightly modified for soil sampling. The soil samples were collected at a depth of 30 cm from the surface of the soil after removing surface organic matter. A single sample for each field was made by putting together all random soil samples of each site.
Each sample contains 25 g of soil, kept in sterile polythene bags, and brought to the laboratory.
Table 1. Economically important plants susceptible to Verticillium wilt
Category Susceptible plants References
Field crops Tobacco, Cotton, Alfalfa (Land, Lawrence, & Newman, 2016; Thomson &
Mcleod, 1959)
Vegetables Potato, Radish, Tomato Cabbage, Cucumber (Berlanger & Powelson, 2000) Fruits Strawberry, Watermelon, Grapes (McCain, Raabe, & Wilhelm, 1981) Trees Olive, Oak, Maple, Walnut (McCain, Raabe, & Wilhelm, 1981)
Samples Preparation and Culturing
For the detection assay of Verticillium, the soil dilution plate method was used (Waksman, 1922).
From the original field samples, 1 g of soil was diluted in 9 ml of sterile distilled water, and two 1:10 serial dilutions were made for each field. Culture plates were prepared by pouring 15 ml of Potato Dextrose Agar (PDA) media (PDA; composition per liter sliced potato 200 g, glucose 20 g, agar 10 g, and ampicillin at 100 µg/ml) into each petri dish.
A total of 100 µl of each soil dilution was plated on PDA (Dhingra, 1985). Three plates per field were prepared, one from the original sample and two from dilutions 1 and 2. One plate was taken as a negative control with no cultured sample. The plates were sealed with parafilm tape and labelled with field names and culturing dates. All the Petri dishes were incubated at 26°C for 7 days.
Isolated colonies on the plates were subsequently subcultured on PDA media to obtain pure colonies. This was done by streaking each colony on the plates with a pipette tip. The pipette tip was then dipped and streaked in an Eppendorf tube containing 50 µl of sterile distilled water. This suspension of spores was plated on the center of PDA plates. The same procedure was followed for each colony. The plates were allowed to dry, sealed with parafilm, and incubated at 26ºC for 7 days.
Inoculum from the margins of 7 days old culture of the isolates was observed under the light microscope. Isolates were identified by observing the morphological characteristics of the fungus based on characters described by Domsch, Gams,
& Anderson (1980). Slides were prepared from the pure culture of the isolates by the following procedure. A small drop of water (40 µl) was poured on the microscopic slides. Colonies on the plates were streaked with pipette tips and dipped in a water drop on the slides. Slides were gently covered with coverslips and viewed under a microscope.
Spores Counting and Colony Diameter
For spores counting, the conidial suspension was prepared from 7 days old culture growing actively on PDA. Conidial suspension of isolates was prepared by adding 3 ml distilled water to plates and scratching gently with a sterile bent glass rod.
The plates’ water-containing spores were collected and transferred to Eppendorf tubes. The spores’
suspension was diluted at least 1/10 by taking 10
µl of the suspension in 90 µl distilled water. Spores were counted by placing 20 µl of spore suspension on the hemocytometer slide and observed under the light microscope. Spores were calculated using the equation 1:
Spores/ml = (n) × 104 (df) ...
.1)
Where: n = the average spore count per square of the four corner squares counted; df = the dilution factor.
Colony diameter was measured by culturing spores suspension at a concentration of 1×107 conidia/ml on the center of PDA and Complete Media (CM). The Complete Media (CM) composition was per liter yeast extract 6 g, casein acid hydrolysate 6 g, sucrose 10 g, ampicillin at 100 µg/ml to find out the growth efficiency of the isolates. The diameter of colonies was recorded on each plate using a ruler with intervals of three days.
Samples Preparation and Culturing
All experiments were carried out in three independent replicates. Means ± standard deviation and significant differences were determined using SPSS 17.0 software (SPSS Inc., Chicago, IL, USA). An Independent sample t-test was used to determine the significant differences with the same software.
RESULTS AND DISCUSSION
Soil samples were collected from tobacco fields in Yarhussain zone II region of district Swabi.
Samples were collected from four different regions of the selected area, Tarkha (2 samples), Saheedan (2 samples), Jalaroona (1 sample), Jagannat (1 sample). Tobacco fields in these regions were noted suspected of Verticillium wilt during the previous year’s investigation (Fig. 1). Numerous studies on soil fungi show that the fungal flora of a region depends on the native soil. Some fungi are ubiquitous, while others are present only in particular habitats (Naranjo‐Ortiz & Gabaldón, 2019). The soil texture, surface vegetation, plenty, nature of the organic content of the soil, and other soil and climatic conditions significantly affect the dissemination of these organisms (Du et al., 2022;
Saravanakumar & Kaviyarasan, 2010). The soil texture of these regions is different, as shown in Table 2.
Table 2. Processed soil samples to isolate Verticillium dahliae using PDA media
Samples Origin Soil texture
2 Field Z Tarkha Loam
2 Field B Saheedan Clay loam
1 Field A Jalaroona Sandy
1 Field P Jagannat Sandy loam
Culturing and Subculturing
Growth of different fungal colonies was observed on all the PDA plates after 7 days of incubation. Some plates were observed to have two or more similar colonies, while others were completely different (Fig. 2). Plates from field Z had four white fungal colonies, while plates from all other fields had two white colonies along with others (Table 3). Soil from four areas was assayed to detect and isolate V. dahliae. Out of which, V.
dahliae was isolated from field Z after subsequent subculturing. No growth of V. dahliae was observed on the plates from other fields. However, species of Fusarium and Actinomycetes were detected on field B plates (Fig 3).
Table 3. Fungal isolates of different tobacco fields on PDA media
Plates Color of fungal colonies Field Z White & yellow Field B White, black & green Field A Cottony white and black Field P White, black & green
Table 4 shows the field-wise positive samples of V. dahliae and its percentage. Like other fungal diseases, Verticillium wilt spreads via infected soils or plant material into new cropping areas.
The presence of an early and reliable detection system and/or quantification is essential to control the disease’s spread and decrease the associated losses to different crops (Sankaran, Mishra, Ehsani, & Davis, 2010). Many scientists have long used the soil dilution method (Waksman, 1922) in assessing soils for their qualitative and quantitative fungal flora (Rao, 1970). Rao (1970) also isolated species of fungi imperfecti from soil using the same method.
Fig. 1. Symptoms of Verticillium wilt on tobacco plants: (A) Vascular discoloration and chlorosis, (B) Complete wilted plant, (C) Foliar wilting
Table 4. Field-wise representation of positive samples of Verticillium dahliae and its percentage
Fields Positive cultures Percentage
Tarkha 2/3 66.6%
Shaheedan 0/3 0%
Jalaroona 0/3 0%
Jagannat 0/3 0%
Colonies of V. dahliae were isolated from soil samples by Lopez-Escudero & Blanco-Lopez (2005). Isolation of V. dahliae is challenging because Verticillium spp. grow slowly in culture media as compared to other fungi. Most of which overgrow on the plates, especially Fusarium spp. before V. dahliae can be morphologically detected and identified (Ausher, Katan, & Ovadia, 1975; Lopez- Escudero & Blanco-Lopez, 2005). Fusarium spp.
was detected on the plates from field B, it may be associated with the wilting of tobacco plants in that field, or it may have masked the growth of V. dahliae.
In some cases, root damage or soil-borne pathogens other than Verticillium, such as Fusarium spp., also results in symptoms similar to Verticillium wilt (Maurer, Radišek, Berg, & Seefelder, 2013).
Begum, Haque, Mukhtar, Naqvi, & Wang (2012) investigated vegetable growing areas of Pakistan for disease incidence of bacterial wilt (BW) caused by Ralstonia solanacearum. They also reported the presence of several other diseases with wilting of solanaceous crops, i.e., Verticillium and Fusarium wilt, bacterial blights.
Morphology of V. dahliae
Isolates on PDA were identified by observing cultural and morphological characteristics under a microscope. Isolates were examined for the color of colonies, conidia morphology, and formation and shape of microsclerotia. Isolates with white colony color, oval spores, septate mycelium with phialides, and oblongata dark melanized structure were identified as V. dahliae. These characteristics were similar as described by Berlanger & Powelson (2000), and Hawksworth & Tallboys (1970). The morphological characteristics of the species may be highly variable and depend upon the incubation conditions and culture media. This variability sometimes hindered the identification of Verticillium species (Jabnoun-Khiareddine, Daami-Remadi, Barbara, & El Mahjoub, 2010).
Fig. 2. Morphology of different fungal isolates: (A) Field 1 fungal isolates (B) Field 2 (C) Field 3 (D) Field 4
Fig. 3. Isolated fungal species from soil: (A) Verticillium dahliae, (B) Fusarium (C) Actinomycetes
Conidial morphology
The isolates of V. dahliae were further characterized by observing the morphology and size of conidia under a microscope. Conidia were oval, unicellular, and hyaline, borne singly on conidiophores (Fig. 4). Conidia are produced at the phialides tip. These characteristics were also described by Jabnoun-Khiareddine, Daami-Remadi, Barbara, & El Mahjoub (2010), and Isaac (1967).
Morphology of Mycelium
The isolates of V. dahliae had erect, totally hyaline, septate, and dense mycelium/ hyphae with two to four phialides at each node of verticillate conidiophores (Fig. 5). The phialides with heads of conidia were borne directly on long horizontal hyphae at uneven intervals. Based on this typical mycelium morphology single isolate of V. dahliae was identified on the plates from the field Tarkha, Yarhussain region of Swabi. Similar characteristics were described earlier (Jabnoun-Khiareddine, Daami-Remadi, Barbara, & El Mahjoub, 2010;
Smith, 1965). Microsclerotia act as the main infective propagule under normal conditions and remain dormant in soil and plant debris for an
extended period. The microsclerotia spread by irrigation water, rain, animal and human activities, agriculture tools, and machines. The parasitic phase of Verticillium begin when microsclerotia is stimulated by root exudates of host plants (Montes- Osuna & Mercado-Blanco, 2020).
Morphology of Microsclerotia
A two-week-old culture of V. dahliae on PDA media was observed for the presence of resting structure, microsclerotia. Microsclerotia of the isolate were dark brown to black and were formed on single hyaline hyphae (Fig. 6). These microsclerotia were very irregular, having subglobose to elongate shape. The culture medium used to grow fungus influences the morphology of microsclerotia.
Several studies indicate that the morphology of V.
dahliae microsclerotia produced on different culture media might be connected to the pathogenicity of the isolate (Lopez-Escudero & Blanco-Lopez, 2005). Defoliating isolates of V. dahliae on water agar produced a mixture of rounded and elongated microsclerotia, while non-defoliating isolates only produced rounded microsclerotia (López‐Escudero, Roca, Valverde‐Corredor, & Mercado‐Blanco, 2012).
Fig. 4. Spores morphology of Verticillium dahliae: Spores were collected by adding sterile distilled water to the plates and gently scraped with a sterile loop. The spores were then observed under a microscope at 40
× magnification, and pictures were taken. (A) Oval and unicellular conidia (B) Specialized fungal hyphae with conidia.
Fig. 5. Mycelial morphology of Verticillium dahliae: Sterile distilled water was added to the 7-day-old culture and was gently scratched for the collection of mycelia and observed under the light microscope at 40 × magnification (A) Verticillate arrangement of phialides on conidiophore (B) Hyaline, septate, and multinucleate mycelium
Fig. 6. Observation of microsclerotia under microscope: (A) Irregular subglobose shaped microsclerotia (B) Dark brown color microsclerotia on single hyphae
The dilution of spore suspension was applied on the hemocytometer slide and observed under a microscope to determine viable spore count. The spores count for V. dahliae isolate was 3.61 × 108 conidia/ml. Different types of variations arise in the process of spores counting. Variability also arises among subsamples from a single culture. While counting spores using a hemocytometer slide, variability arises due to differences among the results of individuals counting squares and sides.
Spores counting are essential to produce inocula for testing the efficiency of the growth medium and pathogenicity of the fungus.
The growth efficiency of V. dahliae was tested by culturing a known concentration of conidial suspension on PDA and CM media. Colony diameter was measured on both media with an interval of three days using a ruler. Fig. 7 and Fig.
8 show the colony diameter of V. dahliae on PDA and CM media on different days post-culturing. The average colony diameter of V. dahliae after nine days of incubation on PDA and CM media was 6.55 cm and 5.5 cm, respectively. The results indicate that V. dahliae growth rate based on measuring the colony diameter is higher on PDA than CM media.
Fig. 7. Colony diameter of Verticillium dahliae on PDA media with three days interval
Fig. 8. Colony diameter of Verticillium dahliae on CM media with three days interval
CONCLUSION AND SUGGESTION
The investigated areas were the significant sites of tobacco products from which soil fungal flora data was unavailable before this study. The investigation was carried out with the aim of isolation and morphological characterization of V. dahliae from tobacco fields. Soil samples from suspected tobacco fields were collected and assayed using the plate dilution method. V. dahliae was isolated from one field (Tarkha, Yarhussain region of Swabi) out of four.
However, a specie of Fusarium and Actinomycetes was also isolated from the Shaheedan, Yarhussain region of Swabi. Isolates of V. dahliae were identified based on its colony characteristics, i.e., spores morphology, mycelial structure, and microsclerotia observation. Isolate with white colony color, oval spores, septate mycelium with phialides, and oblongata dark melanized structure were identified as V. dahliae. This research is the first report on Verticillium biology from the field of Swabi. Based on this study observation, the main goal of future research should be the molecular characterization of isolated V. dahliae. The isolate should also be analyzed for its pathogenicity on tobacco plants. Verticillium wilt has become a significant disease of tobacco during the past years. As no effective control strategy or fungicide is available, it is suggested to use resistant cultivars and avoid fields previously used for susceptible crops (crop rotation). Consequently, this study provides valuable information for tobacco growers and agricultural organizations to apply suitable control strategies against the phytopathogenic fungus V. dahliae.
ACKNOWLEDGEMENT
This work was supported by a grant (SRGP) from Higher Education Commission (HEC) of Pakistan (21-1821/SRGP/R&D/HEC/2018).
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