Abstract: In 2007, date palm trees surveyed for different symptoms on leaves and fruits in Upper Egypt. The samples were collected from symptomatic rachis, fronds and fruits in four localities (El-Minia, Assiut, Sohag and Qena Governorates). The major diseases noticed were leaf spots, off-shoot decline, black scorch, leaf basal rot and fruit spots. Seven different fungal species were isolated from date palm exhibited different symptoms. The results of pathogenicity test proved the ability of Thielaviopsis paradoxa, Alternaria alternate and Negrospora oryza to infect date palm rachis, fronts and fruits, respectively. Helminthosporium sp. and Epicocum nigrum were associated with the leaf spots. E. nigrum was associated with off-shoot decline, black scorch and leaf basal rot, while Fusarium proliferatum and Aspergilus niger were associated with fruit spots. Antagonistic effect of E. nigrum against A. alternate and T. paradoxa was examined by dual culture technique which leads to clear inhibition zone. Calcium chloride (CaCl2) and E. nigrum filtrates effectively controlled the leaf spot (as major disease) when applied at suitable time (when disease severity was low).
INTRODUCTION
Date palm (Phoenix dactylifera L.) is considered one of the most important fruit crops in Egypt. The cultivated area is about 14 million trees (FAO, 2009). It is cultivated all over the country especially in New Valley, Aswan, Sinai, Sharkiya, Beheira, Kafr El-Sheikh and Giza Governorates. There are various economically important diseases infecting date palm trees in all date palm growing countries (Anonymous, 1993; Saadi, 1992; Zaid et al., 2002). Among these diseases, bayoud caused by Fusarium oxysporum f. sp. albedinis (Chakroune et al., 2008), leaf spot caused by Alternaria spp. and Mycosphaerell tassiana (Abdalla et al., 2001; Livingston et al., 2002) inflorescence rot caused by Mauginiella scaetae (Michael and Sabet, 1970) or T. paradoxa (Abdullah et al., 2005), black scorch caused by Thielaviopsis paradoxa (Mubarak et al., 1994; Suleman et al., 2001; Al-sharidy and Molan, 2008), leaf blight caused by Glomerella cingulata (Al-Rokibah, 1991) and off-shoot decline caused by Chalara paradoxa (Molan et al., 2004). According to available literatures, no previous data was published on date palm fungal diseases in Upper Egypt. So, this study was carried out to evaluate the distribution and incidence of various fungal diseases on date palm major growing areas of Upper Egypt. On the other hand, the study was conducted to evaluate of E. nigrum filtrates and CaCl2 in control leaf spot disease.
MATERIALS AND METHODS
Survey of diseases incidence: Surveys were conducted on date palm trees of all ages in El-Minia, Assiut, Sohag and Qena Governorates in 2007 season. Each site was inspected once a month from March to October. Percentage of infection with leaf spot, black scorch, leaf basal rot, fruit rot and off-shoot decline was recorded. Percentage of infection was calculated according to the following formula:
Isolation and identification of causal fungi: Random samples of diseased rachis, fronds and fruits were collected from date palm off-shoots in surveyed area for isolation. Collected samples were washed with tap water then tissues from the margins of lesions on rachis, fruits and frond were surface sterilized with 2% sodium hypochlorite solution for 3 min, rinsed in sterile distilled water, allowed to dry and plated on Potato Dextrose Agar (PDA) at 30°C for 8 days. The developed fungal colonies were purified using single spore isolation technique according to Baudion (1988). The purified fungi were identified according to fungal morphological characteristics as described by Barnet and Hunter (1998) and Moubasher (1993) and then confirmed in Plant Pathology Department, National Research Centre (NRC), Dokki, Egypt. Frequencies of the isolated fungi from date palm parts were calculated according to the following formula:
Pathogenicity test: Inoculation was carried out by techniques used by Hooker (1957) on date palm (Saidy var.). A 8 mm in diameter and 5 mm deep wound (hole) was made at the center of off-shoot rachis by using sharp flamed needle and inoculated with a PDA disc (8 mm in diameter) which was cut from a 10 days old culture plate of Helminthosporium sp., T. paradoxa, E. nigrum, F. proliferatum, N. oryza and A. niger. Agar discs which were containing conidia, chlamydospores and mycelial fragments, were inserted into the whole cuts, covered with small pieces of wet, sterile cotton and covered with polyethylene bags to maintain high relative humidity necessary for infection according to Sultan and El-Hussieni (1999). Fruits and fronds were pin prick and sprayed with the fungal suspensions. Rachis, fronds and fruits free from fungi were used for control test and ten replicates were used in each test. One month after inoculation, the disease severity index (damage necrotic or dark-brown area around the point of infection) was calculated using a scale (0-5), where 0: No apparent infection, 1: 1-10% damage necrotic or dark-brown area around the point of infection, 2: 11-25%, 3: 26-50%, 4: 51-75% and 5: 76-100% according to Baudion (1988). Re-isolation was carried out from infected tissues and the isolated fungi were compared with the original cultures used.
Control of leaf spot caused pathogen and disease
Antagonistic effect of E. nigrum, in vitro: The antagonistic
effect of E. nigrum isolate against A. alternata and T. paradoxa
(as main pathogens) was tested by dual culture method according to Hozarika
and Das (1998). E. nigrum was inoculated 48 h earlier before the
fungi tested. After 5 days of inoculation at 28±2°C on PDA medium,
the radial growth of the fungi tested and inhibition zones were measured.
Effect of CaCl2 and E. nigrum exudates in controlling leaf spot disease under field conditions: The experiment was conducted in date palm trees (5 to 8 years old) off-shoot (Saidy var.) growing on privet farms located at Qena Governorate. All palm trees used in this study showed high disease severity (as a nature infection) at the start of the experiment. The 3rd whorl leaves from the bottom of tested trees were selected and labeled. Three trees were used as a replicates for each treatment. E. nigrum culture filtrate (diluted to 50%) and CaCl2 (0.5 g L-1) were used. Treatments were sprayed once a month starting from March to October 2009. The disease severity ranged from 3.1-6.7 at the initiation of spray. At starting November 2009, another set of leaves, the 6th whorl from the bottom was selected and labeled for observation. Less than 2.6 disease severity was observed at the start of spray which continued until October 2010. Five leaflets from each labeled leaf were selected at equidistant and disease severity was calculated using the following formula (James, 1971):
RESULTS
Survey: The main symptoms noticed during survey were leaf spots, black scorch, leaf basal rot, off-shoot decline and fruit spots. Leaf and fruit spots had the highest disease incidence, compared with the other disease symptoms in most regions (Fig. 1). Black scorch was found in Assiut and Sohag Governorates (ranged from 1.8-2.3%). Fruit spots found to be the highest symptoms in three over four surveyed Governorates (ranged from 3.2-6.4%). El-Minia regions showed lowest symptom incidence, whereas, leaf spots only was noticed 2.2%.
Field symptoms
Leaf spots: Symptoms appeared as small scattered, irregular, brown
to black (about 3-7 mm). The spots appeared on the upper and lower surface of
rachis and fronds (Fig. 2a).
Off-shoot decline: The symptoms appeared firstly on one or more of the inner leaves then appeared on the outside ones. It started on fronds as light brown blights with dark brown edges (Fig. 2b1, b2) then extended to the rachis midrib on both surface as a black color (Fig. 2b3) causing death of midrib followed by their drooping down.
| Fig. 1: | Survey for symptoms and incidence of some diseases in date palm growing Governorates on Upper Egypt |
| Fig. 2(a-e): | Different symptoms occurred in date palm tress as nature infection. (a) Leaf spots in rachis, (b1) Off-shoot decline in off-shoot; (b2) Fronds and (b3) Rachis, (c) Black scorch in leaf, (d1) Leaf basal rot at the base of leaves, (d2) Central leaf cluster die and (e) Fruit spots |
Black scorch: Symptoms appeared firstly as grayish discoloration at leaves which turned into black discoloration followed by dwarfing and malformation (Fig. 2c).
Leaf basal rot: The symptoms appeared as brown to dark brown rot at the base of leaves (Fig. 2d1). The central leaf cluster might die before the older leaves and can easily detached (Fig. 2d2).
Fruit spots: Symptoms appeared as circular, black and stone spots in all parts of the fruits (Fig. 2e).
Isolated fungi and pathogenicity: Seven fungi associated with date palm disease symptoms were identified to species. The majority of isolates were obtained from leaves exhibited leaf spots, black scorch, off-shoot decline and fruit spots. The percentages of the predominance of A. alternate from samples exhibited leaf spot and basal rot (43.1-33.3%, respectively). T. paradox (Table 1, Fig. 3) was the most frequently isolated fungi from samples exhibited black scorch and off-shoot decline symptoms (52.7-37.4%, respectively). N. oryza was the most dominant fungi isolated from fruit spots (27.5%). Among seven fungal isolates obtained from date palm parts exhibited various disease symptoms, A. alternate, T. paradoxa and N. oryzae were pathogenic Table 1 and Fig. 3. The other isolates, Helminthosporium sp., E. nigrum, F. proliferatum and A. niger were non-pathogenic. Helminthosporium sp. and E. nigrum were associated with leaf basal rot and leaf spots, E. nigrum with black scorch and off-shoot decline, also F. proliferatum and A. niger with fruit spots.
Control of leaf spot disease
Dual culture antagonism: The antagonistic effect of E. nigrum
against the most frequent fungi (A. alternate, T. paradoxa) was
studied. The obtained results demonstrated that the radial growth of the two
fungal species was significantly inhibited by E. nigrum. The mean inhibition
zone was 0.82 and 0.66 for A. alternate and T. paradoxa, respectively.
Application of CaCl2 and E. nigrum exudates against date palm leaf spot disease: When the spray was initiated in March 2009, the disease severity on the 3rd whorl leaves ranged from 3.1-6.7. Although, the E. nigrum exudates and CaCl2 were sprayed every month until October 2009, the disease didn’t decrease but increased gradually (Fig. 4). The spray was continued and new disease that developed on the 6th whorl leaves was recorded from November 2009 to October 2010. The disease severity on the 6th leaves ranged from 1.2-2.6% at time of the first spray. In two treatments, the disease severity decreased gradually. The disease severity didn’t vary significantly between two treatments, although, the differences between the treatments and control were highly significant (Fig. 5-6).
| Table 1: | Frequency and pathogenicity of fungal pathogens isolated from date palm |
| Fig. 3(a-c): | (a) Microscopic photograph of T. paradoxa isolate showing microconidia: which were hyaline and cylindrical form, also, chlamydospores: have dark brown with thick walls, (b) Pathogenicity test for A. alternate on fronds and (c) T. paradoxa on rachis |
| Fig. 4: | Disease severity of leaf spot disease on the 3rd whorl leaves of date palm from March to October 2009 after CaCl2 and E. nigran exudatis spraying |
| Fig. 5: | Disease severity of leaf spot disease on the 6th whorl leaves of date palm from November 2009 to February 2010 after CaCl2 and E. nigran exudates spraying |
DISCUSSION
Fungi are known as the most pathogens on date palm trees (Zaid et al., 2002). The present study showed limited spread of fungal diseases on date palm trees in the surveyed area because of the dry conditions and high temperature in surveyed regions. The results indicated that leaf and fruit spots are widespread. High incidence of two diseases may be due to the absence of control measures in Upper Egypt. Diseases are common on similar conditions in Saudi Arabia (Al-sharidy and Molan, 2008), Kuwait (Mubarak et al., 1994), Sultanate of Oman (Anonymous, 1993) and Iraq (Djerbi, 1983; Sarhan, 2001). Despite the widespread of leaf spot disease, the author noticed absence of Graphiola leaf spot, this may be due to the dry climate. Black scorch and leaf basal rot are low spread. A. alternate, T. paradoxa and N. oryza were the most predominant fungi isolated from date palm trees showed visible symptoms. It was pathogenic 13-19 days after inoculation, confirming Koch’s postulates. Similar results were reported by El-Meleigi et al. (1986), Sarhan (2001), Al-sharidy and Molan (2008) and Abdullah et al. (2009).
| Fig. 6: | Disease severity of leaf spot disease on the 6th whorl leaves of date palm from March 2010 to October 2010 after CaCl2 and E. nigran exudatis spraying |
There has been evidence in recent years of the role calcium in plant nutrition relative to lessening the impact of Pythium, Sclerotium, Botrytis and Fusarium at many crops. Calcium has important roles in the integrity of cell walls which infers a resistance mechanism to reduce penetration of fungal pathogens. Among the fungi that we isolated, E. nigrum selected because of ability to inhibit growth T. paradoxa and A. alternate on PDA through production of diffusible metabolites. The inhibition of T. paradoxa and A. alternate by E. nigrum is similar to the inhibition growth of Phytophthora erythroseptica and T. paradoxa by Trichoderma virens and T. longibrachiatum, respectively (Etebarian et al., 2000; Sanchez et al., 2007).
Although, the E. nigrum exudates and CaCl2 were sprayed every month until October 2009, the disease didn’t decrease but increased gradually. This implies that treatments used were not effective in controlling the disease when the disease severity was high (above 3.1) at the initial time of application. From the results obtained, it can be concluded that spraying E. nigrum exudates or CaCl2 before the initiation of the symptoms can reduce the incidence of leaf spot disease.