Isolation and identification of the different pathogens of date palm diseases

Introduction

The history of date palm cultivation throughout the world dates back approximately to 4000 B.C. or earlier. Date palm (Phoenix dactylifera L.) is a monocotyledon, dioecious plant and one of the most cultivated palms around the world (Abass, 2013) [1] . Date palm trees are cultivated in different regions worldwide, especially in Middle East, North Africa, Central and North America, Southern Europe, Pakistan, and India (Zaid, 2002) [23] . Mainly the cultivation of date palm is aimed for its fruits which are highly nutritious and also have many medicinal uses. Date palm fruits are eaten as raw dates (fresh fruits), dry dates (chhuhara) and soft dates (pink khajoor). Apart from the fruits, other parts of the date palm also have traditional as well as economic usage. The flesh of dates contains a high percentage of carbohydrate (44-88%), protein (2.3-5.6%), dietary fibre (6.4-11.5%) and fat percentage reach to (0.2-0.5%), including 14 types of fatty acids (Alkahtani et al.,2011) [4] . In India, climate of Gujarat, Rajasthan and some areas of Punjab is suitable for the production of date palm. The commercial cultivation of date palm occurs mainly in the western border of India and the coastal belt of Kutch area of Gujarat which contributes about ninety per cent of the total date production in India Several fungi attach date palm causing root rot, wilt and decline diseases (Abdalla et al., 2000) [2] . Several soil borne fungi were recorded as causal pathogens of date palm, viz., Fusarium oxysporum; F. solani; F. moniliforme; F. semitectium; F. equiseti; Phoma sp.; Chaetomium sp.; Alternaria sp.; Cladosporium sp.; Macrophomina phaseolina; Thielaviopsis paradoxa; Diplodia phoenicum; Phomopsis phoenicola, and Rhizoctonia solani (Abdalla et al., 2000; El-Zawahry et al., 2000; Sarhan, 2001; Suleman, et al., 2001; El-Morsi, 2004) [2,13,20,22,12] . The most common disease of date palm was Bayoud disease caused by Fusarium oxysporum f.sp. albedinis. This disease is the most serious especially in Moracco and Algerias (Elarosi, 1989; El-Hassni et al., 2005) [8,10] .

Fusarium monliforme and F. solani were found associated with declined date palm trees in Egypt. The dominant fungi associated with date palm foliar diseases are: Helminthosporium sp. and Alternaria sp. (leaf spots) and Botrydiplodia theobromae (Basal rot), (Al-Sharidy and Molan, 2008) [5] .

Materials and methods

The investigation entitled "Identification of diseases in date palm (Phoenix dactylifera L . ) . " w a s c a r r i e d o u t i n t h e Department of Plant Pathology, Chimanbhai Patel College of Agriculture, Sardarkrushinagar Dantiwada Agricultural University, Sardarkrushinagar during 2020-21. The materials used and methodology followed for the isolation of pathogen, their pathogenicity, morphological and cultural characteristics, under in vitro condition are describe d as under.

Isolation and identification of the different pathogens of date palm diseases Collection of diseased samples, isolation and purification of pathogen.

The diseased samples of date palm showing typical symptoms were collected from Date palm Research Station, Mundra, Agroforestry Research Station and Horticulture Instructional Farm in S. D. Agricultural University, Sardarkrushinagar. The infected samples were brought to the laboratory for further studies. The pathogens were isolated from diseased specimens using the "tissue isolation technique". The typically infected leaves and root portion were washed in running tap water and then air dried. The diseased part along with some healthy portion were cut into small pieces with the help of sterilized razor and again washed with sterilized water. These pieces were kept for one minute in one per cent sodium hypochloride solution for disinfection. These pieces were washed thrice in sterilized distilled water to remove residues of sodium hypochlorite. The pieces were transferred aseptically onto sterilized Potato Dextrose Agar (PDA) medium in Petri-plates. The Petri-plates were incubated at 27 ± 20 °C temperature for seven days in BOD incubator. Purification of spore forming fungal culture was done by using single spore suspension method. In this method, water agar (2%) was poured into Petri-plates of 90 mm diameter and allowed to solidify. The spore suspension was prepared by diluting 5 mm sized mycelial disc into 5 ml sterilised distilled water. 5 μl of spore suspension was transferred onto the water agar using a micro pipette. Spore suspension was spread uniformly over the water agar by streaking with Lshaped inoculating needle/spreader. The plate was incubated at 27±1 °C for 2 to 3 days. Germinating conidia on the culture plate were located using low power microscope. A portion of the media with an isolated germinating conidium was excised from the water agar medium using a sterilized needle and was transferred to PDA medium for incubation at 27±1 °C temperatures (Niemeyer and Andrade, 2015) [17] . Purification of non-spore forming fungal culture was done by using hyphal tip method. Culture of the test pathogen was grown on water agar (2%). Medium was poured in less quantity to the 90 mm diameter Petri-plate so as to limit the growth of the test pathogen. The individual hyphal tip from the margin of sparsely grown culture was excised using a sterilised needle and transferred to Petri-plates containing PDA medium (Leslie and Summerell, 2006) [15] . The plates were incubated at 27±1 °C temperatures for further growth.

Identification

Identification of pathogens grown on PDA medium were examined visually as well as microscopically for cultural and morphology characters. The cultural characters were recorded right from initiation of growth up to 15 days. The morphological characters viz., length and width of spores, mycelium etc, were measured under high power magnification from 10 days old culture of pathogens and were compared with those given in the literature or manual. The photomicrographs of the same were also be taken. The identification was based on taxonomic identification keys.

To prove the pathogenicity Root dip inoculation method of root infecting pathogen

Date palm seedlings grown in autoclaved sand filled in pots were uprooted, and washed in running tap water and clipped from the distal one-third root system. These seedlings were dipped for 10 minutes in the spore suspension (1×10 6 spores/ml). Inoculated seedlings were then transplanted into earthen pots filled with autoclaved soil. The seedlings treated without inoculation were kept as control. The test was conducted in three replicates.

Foliar spraying of spore suspension of leaf infecting pathogen

The pathogenicity of the foliar microbes was tested on the leaves of date palm seedlings grown in the earthen pots (30 cm diameter) filled with sterilized soil and FYM in the ratio of 3:1. Date palm seedling were grown in earthen pots, covered with plastic to avoid any air-borne infection. The pots were labelled, watered gently and arranged in line. The pots were watered up to saturation in the morning. The leaves were surface sterilized with 1 per cent sodium hypochlorite (NaOCl) solution and washed thoroughly with sterilized distilled water to remove the traces. Three months old date palm seedlings were used for inoculation. The spore suspension of the microbes were sprayed on the leaves in the evening with newly fresh inoculum. The control pots without inoculation were sprayed with sterilized distilled water. The inoculated and uninoculated pots were covered with polyethylene bags for 48 hour to provide high humidity. The observations on the disease development on leaves were recorded periodically from the initiation of the disease. Re-isolation were made from the artificially inoculated plants showing typical leaf spot symptoms by tissue isolation method and the identity of the fungus were confirmed as per the original description. The culture obtained by re-isolation was transferred on PDA slants for comparison with the original culture and further investigations.

Morphological and cultural studies of isolated pathogen from date palm

All the isolates established and maintained on PDA were studied for their cultural and morphological characters. In the study of solid medium, the discs were inoculated in the centre of 90 mm Petri-plate containing 20 ml of PDA medium. Observations on fungal radial growth, colony character, sporulation and pigmentation were recorded after 7 days of incubation. The radial growth was determined by measuring the colony diameter in mm along the two diagonals passing through the centre of colony and the average were worked out. For recording sporulation in each isolate, the spore suspension was prepared by randomly cutting five uniform discs (4 mm) of fungal colony, raised on solid media, putting it and shaking it well in 5 ml of water in 50 ml flasks. The droplet of the spore suspension was placed on a clean glass slide and spores were checked at low power (10x) field of microscope. The sporulation were counted with the help of haemocytometer, whereas length and breadth of conidia were measured by ocular micrometre. Colonies of fungus emanated from the tissues were subjected for isolation on PDA medium. Growing hyphal tips from the margin of these white colonies were transferred to fresh PDA medium and incubated at 27 ± 1°C. The culture so obtained was purified using different techniques and used for further investigation. Results obtained further suggested that Fusarium sp. and Macrophomina phaseolina caused root rots diseases and Alternaria sp. caused leaf spot diseases on date palm. Ahmed (2018) [3] mentioned that some soilborne fungal diseases viz., Fusarium solani, F. oxysporum, Rhizoctonia solani and Macrophomina phaseolina affect the roots of the date palm. Elhassan (2016) [9] isolated Fusarium solani causing the wilt disease in date palm trees. However, Baraka et al. (2011) [7] isolated Fusarium sp. most frequently from the root rot on date palm. Leaf spot diseases are the most widely spread foliar disease in date palm. Similarly, Ammar and El-Naggar et al. (2011) + isolated leaf spot causing Alternaria alternata from the leaves of date palm.

Results and Discussion

Identification of pathogen

The purified culture of the fungal isolates responsible for the wilt, root rot and leaf spot pathogen on date palm seedlings were examined for initial identification of the fungus. Based on the growth pattern in Petri-plates as well as mycelial and conidial structures under a microscope, the fungal culture was identified. The identification of the fungus isolate was made based on in vitro culture characteristics on Potato Dextrose Agar (PDA) and morphological features (micro, macro conidia and chlamydiospores shape and size) under the microscope (Ellis, 1971) [11] . Based on cultural characteristics, pathogenicity and biological performance of the isolated fungus, the causal agent was identified as Fusarium sp. (Lesile and Summerell, 2006) [15] constituting by that the first report of Fusarium wilt disease on date palm in the Sudan (Elhassan et al., 2016) [9] . The isolates were classified based on their morphological characters on PDA plates. Alternaria isolates grew rapidly and produced colonies with different shapes. The colony was flat and was covered with grayish, short, aerial hypha. Its surface was grayish-white at the beginning which later darkens and becomes greenish-black or olive-brown with a light border. The reverse side was typically brown to black due to pigment production Hypha were septate and brown. Conidia and proconidia were appeared brown with an elongated beak-like apical cell, often in chains and sometimes solitary (Badawy et al., 2019) [24] . Based on this information, the fungal cultures were identified Fusarium s p . , Macrophomina phaseolina and Alternaria sp. and was considered to be cause infection on the root and leaves of date palm seedlings.

To prove the pathogenicity Pathogenicity test was attempted by different inoculation method (root dip inoculation method and spore suspension method) for different pathogens on healthy date palm seedlings grown in pots. Plants were incubated in the mist house, where relative humidity (80%) and optimum temperature (27 ± 2 °C) were maintained for the further development of symptoms.

Root dip inoculation method of root infecting pathogen

Root dip inoculation method of root infecting pathogens were performed to confirm the pathogenicity of the test pathogens. The seedlings grown in autoclaved sand filled in pots were uprooted, and washed in running tap water, and clipped from the distal one-third root system. These seedlings were dipped for 1 hr in the spore suspension (1×10 6 spores/ml). Inoculated seedlings were then transplanted into earthen pots filled with autoclaved soil. The seedlings without inoculation were kept as control. In Fusarium sp. and Macrophomina phaseolina p r o v e d pathogenicity by this method. Symptoms on seedlings infected with Fusarium sp. was observed on the leaves of the inoculated seedlings of the date palm resulted in drying and general death of the seedling. In Marophomina phaseolina mortality of plant was observed. in the early study, Elhassan (2016) [9] tested pathogenicity against Fusarium oxysporium caused wilt disease on date palm. Two months old plants were inoculated with 2 ml of inoculums suspension, the early symptoms observed on the leaves of the inoculated seedlings of the date palm appeared as brown spots, which later resulted in drying and general death of the seedling. Similarly, Purwati et al. (2008) [19] compared different inoculation methods of Fusarium oxysporum f. sp. cubense (Foc) in Abaca. In the previous literature Sukanya et al. (2016) [21] tested seedling roots were dipped for 2 min in M. phaseolina spore suspension of 10 5 spores per ml and these seedlings were kept for germination. The early mortality of the plant was observed Foliar spraying of spore suspension of leaf infecting pathogen Pathogenicity test was attempted by spraying the spores on healthy three month-old date palm seedlings grown in pots. Plants were incubated in the mist house, where relative humidity (80%) and optimum temperature (27 ± 20 °C) were maintained for the further development of symptoms.

Results revealed that Alternaria sp. was pathogenic to date palm seedling causing Alternaria leaf spot. After 30 days of incubation, typical symptoms of Alternaria leaf spot on the foliage of artificially inoculated date palm seedling were observed. However, the control plant sprayed with only sterilized distilled water remained healthy and did not produce any kind of symptoms throughout the observation. The fungus was re-isolated from the inoculated leaves and was compared with the original culture of the test pathogen. The same was found identical to that of the original culture, thereby confirming the pathogenicity.

Inoculated Control

Pathogenicity of Alternaria sp.

In early study, Badawy et al. (2019) [ 2 4 ] tested the pathogenicity of foliar pathogens on date palm seedling. The isolated fungus was inoculated via spraying spore suspension results showed that the pathogen is capable of making infection. This can be seen via the increasing length of lesions. Black coloured lesions were noticed in all inoculated plants. Moreover, black spots were noticed on leaves long distance from the inoculated area on leaves' both sides.

Cultural and morphological study of the isolated pathogen from date palm

The cultural and morphological characters of the isolated pathogens from date palms were studied by growing them on potato dextrose agar (PDA) medium at 27 ± 2 °C after 7 days incubation. Macroscopic growth features viz., colony diameter (mm), cultural characteristics, and microscopic growth features, viz., size of spores and septation in spore, micro and macroconidia and chlamydospores on potato dextrose agar medium in Petri-plates were recorded after 7 days of incubation.

Cultural and morphological study of Fusarium sp.

Cultural characters of the isolates were identified as white fluffy colonies which gradually turned orange after ten days of incubation. Orange pigmentation was visible at the bottom side of the Petri-plate within seven days of incubation as well as in the culture incubated at 4 °C. The radial growth of mycelium attained an average size of 90 mm with a circular margin in seven days of incubation. The culture produced aerial mycelium without any undulations.

Microscopic characteristics showed the formation of a fusiform, slightly curved macroconidia, with 3-4 septate. [14] . The fungus produced profuse white aerial mycelium later turns into brown to black on PDA. Black hard sclerotia were formed after 10-15 days at the periphery of the colony. The young hyphae of the fungus were observed to be hyaline, thin-walled light brown to dark brown in colour, and branches arised from the main hyphae at a right angle to parent hyphae with constriction of the point of origin. The colour of the sclerotia was light brown at the beginning which became darker with age and finally brown to black, sclerotia varied in shape irregular or spherical or oval or oblong, measuring from 90-120 × 60-150 µm with an average of 82.5 to 105 µm in size. But in the present study sclerotia on the fungal culture was not observed. The young hyphae of the fungus were observed to be hyaline, thin-walled light brown to dark brown, and branches arose from the main hyphae at a right angle to parent hyphae with constriction of the point of origin. Based on the cultural and morphological character, this fungus had been identified as Macrophomina phaseolina.

Cultural characters of M. phaseolina

Morphological characters of M.phaseolina

Cultural and morphological study of Alternaria sp.

After seven days of incubation, the colony diameter of Alternaria sp. was recorded as 85.00 mm. The growth of mycelia was slightly fluffy, olive greenish in colour with raised margins in a uniform concentric fashion. The microscopic study of Alternaria sp. revealed that the colony was flat and was covered with grayish, short, aerial hypha. Its surface was grayish-white at the beginning which later darkens and becomes greenish-black or olive-brown with a light border. The hyphae were septate and irregularly branched. The average length and width of the spores were 30.39 and 12.18 μm, respectively. Likewise, the average beak length was 3.97 μm with 4-5 transverse septa and 1-2 longitudinal septa Thus, it is confirmed that Alternaria sp. was responsible for the leaf spot on date palm seedlings. Descriptive information on Alternaria sp. was presented by Badawy et al. (2019) [24] who found similar results on cultural characteristics of A. alternata as were recorded in the present study. They recorded that the colony was flat, and was covered with grayish, short, aerial hypha. Its surface was grayish-white at the beginning which later darkens and becomes greenish-black or olive-brown with a light border. The reverse side was typically brown to black due to pigment production. Hypha septate were brown. Conidia and proconidia appeared brown with an elongated beak-like apical cell, often in chains and sometimes solitary. A similar study by Mirhosseini et al. (2015) [16] cultures gave rise to initially white colonies which turned to grayishblack later due to abundant sporulation. Conidia were produced in long chains, pale to light brown, with a beak one to seven transverse and up to three longitudinal septa, and measured 10 to 45 μm long × 7 to 22.5 μm wide. Thus, the results obtained in the present investigation corroborate the findings of the earlier research workers. However,

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