Graphical abstract
Keywords: Anthracnose, Dracaena sanderiana, Colletotrichum dracaenophilum, Lucky bamboo
Abstract
Dracaena sanderiana, of the family Liliaceae, is among the ornamental plants most frequently imported into Egypt. Typical anthracnose symptoms were observed on the stems of imported D. sanderiana samples. The pathogen was isolated, demonstrated to be pathogenic based on Koch’s rule and identified as Colletotrichum dracaenophilum. The optimum temperature for its growth ranges from 25 to 30 °C, maintained for 8 days. Kemazed 50% wettable powder (WP) was the most effective fungicide against the pathogen, as no fungal growth was observed over 100 ppm. The biocontrol agents Trichoderma harzianum and Trichoderma viride followed by Bacillus subtilis and Bacillus pumilus caused the highest reduction in fungal growth. To the best of our knowledge, this report describes the first time that this pathogen was observed on D. sanderiana in Egypt.
Introduction
Lucky bamboo (Dracaena sanderiana hort. ex Mast.) is among the ornamental plants most frequently imported into Egypt. This bamboo is also known as Dracaena braunii [1]. Although the word bamboo occurs in several of its common names, D. sanderiana is actually of an entirely different taxonomic order from true bamboos. In Egypt, lucky bamboo is the most popular indoor plant and is frequently imported and resold in attractive pots. Colletotrichum spp. is an imperfect fungus belonging to the Melanconiales. Members of the genus Colletotrichum cause diseases on a number of host plants. These diseases, often referred to as anthracnose, include strawberry black spot and key lime anthracnose (caused by Colletotrichum acutatum), tomato fruit anthracnose (caused by Colletotrichum coccodes), red sorghum stalk rot (caused by Colletotrichum graminicola), coffee berry disease (caused by Colletotrichum kawahae), bean anthracnose (caused by Colletotrichum lindemuthianum) and many others [2]. Additional species of Colletotrichum with conidia greater than 20 μm have been encountered on living plants of D. sanderiana (lucky bamboo) from China [3]. In Bulgaria and Iran, Bobev et al. [4] and Komaki et al. [5], respectively, provided the first reports that Colletotrichum dracaenophilum infects the stems of potted D. sanderiana plants, causing anthracnose disease. In the United States, Sharma et al. [6] isolated, characterized and tested fungicide treatments to control Colletotrichum spp. causing anthracnose on lucky bamboo, D. sanderiana. They also reported that C. dracaenophilum caused the most severe disease on lucky bamboo, whereas one isolate of the Colletotrichum gloeosporioides species complex was less pathogenic to all Dracaena spp. and varieties tested. In Egypt, during March 2015, anthracnose symptoms were recorded on D. sanderiana plants. Therefore, the objectives of this work were (i) to describe the symptoms of lucky bamboo anthracnose, (ii) to isolate, identify and test the pathogenicity of the causal agent, (iii) to determine the effect of temperature on the growth of the causal pathogen and (iv) to evaluate the effect of certain fungicides and biocontrol agents on the growth of the pathogen.
Material and methods
Isolation and identification of the causal pathogen
In March 2015, disease problems on the stems of imported (Netherlands) indoor lucky bamboo plants (D. sanderiana) were observed. The symptoms were observed during several months after the consumer purchased them from the retail stores located in Giza governorate. More than 50 diseased samples with typical anthracnose symptoms were collected to isolate the pathogen based on Koch’s rule [7]. The obtained fungal colonies were identified according to the Colletotrichum description reported by Sutton [8] and according to Farr et al. [3].
Pathogenicity tests
Pathogenicity was confirmed by fulfilling Koch’s postulates on rooted cuttings of lucky bamboo plants as well as detached stem segments, according to Bobev et al. [4]. Twenty cuttings of lucky bamboo plants were surface disinfected with 1.5% sodium hypochlorite (NaOCl) for 5 min, followed by several rinses with sterile distilled water before being sown in five glass bottles containing 500 mL sterile water. Thirty days later, these bamboo plants were divided into two sets. The stems of the first set were wounded (ten wounds per plant) using a sterile needle at 4 cm intervals. The stems of 5 plants of the first set were inoculated by inserting small mycelial plugs from 10-day-old potato dextrose agar (PDA) cultures of C. dracaenophilum into wounds, which were subsequently covered with Parafilm strips. Pure agar plugs were used to inoculate the wounded stems of the control plants (5 plants). Both inoculated and control plants were kept at 28 ± 2 °C. Anthracnose symptoms were observed visually for sixty days after inoculation. The stems of the second set (5 plants) were injected with 0.5 mL plant−1 of C. dracaenophilum conidial suspension (2 × 106 conidia mL−1) using a sterilized syringe [9]. The injected and un-injected (5 plants) stems of lucky bamboo plants were covered with plastic polyethylene bags for 24 h to provide humid conditions. Anthracnose symptoms were observed visually. In addition, the stems of apparently healthy lucky bamboo plants were cut longitudinally and horizontally into 1–3 cm segments. These segments were inoculated with one drop of C. dracaenophilum conidial suspension (2 × 106 conidia mL−1) after surface disinfection. Five stem segments from each type of longitudinal and horizontal pieces were used as replicates, and the experiment was replicated twice. The rot of the detached stem segments was observed visually.
Effect of temperature on the growth of C. dracaenophilum
Fresh potato dextrose agar (PDA) plates were inoculated with a 5 mm mycelial disk cut with a sterile cork borer from the margin of a 10-days-old colony of C. dracaenophilum. Plates were incubated in an incubator at 5, 10, 15, 20, 25, 30, 35 and 40 °C. The radial growth of C. dracaenophilum was measured in two perpendicular directions at 4, 8, 10 and 14 days after inoculation. Four Petri plates were used as replicates for each combination of temperature and incubation period.
Inhibitor effect of fungicides on the growth of C. dracaenophilum
The inhibition effects of ten different fungicides under different concentrations viz., 0, 25, 50, 100, 200, 300, 400, 500 and 600 ppm against the pathogen were determined. The systemic fungicides were dimethomorph 6% + copper oxychloride 40% (Acrobat Copper 46%), carbendazim (Kemazed 50% WP), flutolanil (Moncut 25% WP), mancozeb (Tridex 80%), metalaxyl M + mancozeb (Ridomil Glod 68%), and thiophanate-methyl (Topsin-M 70% wettable granul (WG) and the protective fungicides were Mancozeb 80% (Dithane M-45), pencycuron (Monceren 25% WP) and thiram + tolclofos-methyl (Rizolex T 50% WP). The Metalaxyl 8% WP + Mancozeb 64% (Tasoline) is systemic and protective fungicide. The inhibition effect was tested using the poisoned food technique described by Uribe and Loria [10]. Four Petri plates were used as replicates for each treatment as well as untreated control. The average radial growth of C. dracaenophilum was measured in two perpendicular directions when C. dracaenophilum reached full growth in the control plate.
Inhibitor effect of biocontrol agents on the growth of C. dracaenophilum
Fungal and bacterial biocontrol agents viz., Trichoderma harzianum, Trichoderma viride, Trichoderma virens, Trichoderma koningii, Pseudomonas fluorescens, Bacillus subtilis, Bacillus megaterium and Bacillus pumilus were obtained from the Plant Pathology Department, National Research Centre (NRC). The inhibitor effect of the fungal biocontrol agents against the growth of C. dracaenophilum was studied using the method described by Bell et al. [11]. Petri plate containing PDA medium was inoculated on one side with a 5 mm mycelial disk from a 7-day-old culture of the test fungus. The opposite side was inoculated with a disc of C. dracaenophilum and the plates were incubated at 28 ± 1 °C. Plates inoculated with a disc of C. dracaenophilum by itself were used as a control. Four replicate plates were made for each test fungus as well as the control. Colony radius of C. dracaenophilum was recorded when the control plates reached full growth. The inhibitory effect of the bacterial biocontrol agents was studied using the method described by Estrella et al. [12]. Petri plate containing PDA medium was inoculated (by streaking) on one side with one loopful from a 48-h-old culture of the test bacterium. The opposite side was inoculated with a disc of C. dracaenophilum and the plates were incubated at 28 ± 1 °C. Plates inoculated with a disc of C. dracaenophilum by itself were used as a control. Four replicate plates were made for each test bacterium as well as the control. Colony radius of C. dracaenophilum was recorded when the control plates reached full growth. The reduction in the growth of C. dracaenophilum was calculated using the formula suggested by Pandy et al. [13] as follows: Growth reduction (%) = [(C − T)/C] × 100, Where: C = Average growth of C. dracaenophilum in control and T = Average growth of C. dracaenophilum in biocontrol agent treatment.
Statistical analysis
Statistical analysis for a randomized complete block design (RCBD) with two factors and interaction terms was performed for all experiments according to Gomez and Gomez [14]. Least significant difference (LSD) values were calculated to test the significance of differences between means according to Steel et al. [15] (Table 1).
Table 1.
Analysis of variance for RCBD.
| SOV |
Table 3 |
Table 4 |
||
|---|---|---|---|---|
| df | P-values | df | P-values | |
| Replication | (r − 1) = 3 | 0.48 | (r − 1) = 3 | <0.01 |
| A (temperature T or fungicide F) | A − 1 = 7 | <0.01 | A − 1 = 9 | <0.01 |
| B (incubation period I or concentration C) | B − 1 = 3 | <0.01 | B − 1 = 7 | <0.01 |
| AB | (A − 1) (B − 1) = 21 | <0.01 | (A − 1) (B − 1) = 56 | <0.01 |
| Error | (AB − 1) (r − 1) = 93 | (AB − 1) (r − 1) = 237 | ||
| Total | (ABr − 1) = 127 | (ABr − 1) = 314 | ||
Results and discussion
Symptomatology and the causal pathogen
The primary symptoms of lucky bamboo anthracnose were pale green yellowish lesions that appeared on the stems. These symptoms extended to the upper and lower internodes, which became yellow. The hard tissues turned soft, the plant showed wilt symptoms, and the entire stem was covered with numerous black globose ellipsoid acervuli with sparse, black setae Fig. 1. The pathogen was isolated and identified as C. dracaenophilum D. F. Farr & M. E. Polm, according to the classification of Sutton [8] and Farr et al. [3]. C. dracaenophilum has been reported in many regions, such as Cyprus [16], China and Kenya [18], Bulgaria, [4], Iran [5], and the United States [17], including south Florida and retail stores in north Florida [6]. This work is the first report of this fungal species in Egypt. The morphological characteristics of the pathogen are shown in Table 2 and Fig. 2. Colonies of the fungus grown on PDA were dominated by pale aerial mycelium. The acervuli on dying stems, numerous in the discolored areas, were arranged concentrically on the stem, forming tiny black spots, 264–382.8 μm in the longest dimension, with septate setae, sparse, scattered in the hymenium, black, 180–295 × 3.75–6.25 μm, straight to slightly curved, becoming narrow and hyaline at the rounded apex, 4–11 septet. Acervuli were also produced on PDA medium. On the plant and in culture, the conidia were hyaline broadly clavate to cylindrical, occasionally slightly curved, and measured 23–33 × 6.6–9.9 μm (28 long × 8.25 width μm). The pathogen can be expected anywhere in the world where infected lucky bamboo cuttings are imported from China [6]. Even in the Netherlands, the cuttings actually come from China. Sinclair [19] and Verhoeff [20] stated that even in the absence of visible symptoms, Colletotrichum spp. may persist on plants as microscopic latent infections consisting of appressoria with limited development of infective hyphae. Sharma et al. [6] made the same observation and reported that lucky bamboo introduced from China might carry C. dracaenophilum, which can induce anthracnose symptoms several months after arrival in the United States. It is not known which environmental factors might trigger the appearance of symptoms. However, Sharma et al. [6] mentioned that anthracnose lesions appeared on non-inoculated stalks of D. sanderiana plants when the irrigation intervals were lengthened. Thus, water stress may trigger symptoms.
Fig. 1.
Natural symptoms of lucky bamboo anthracnose disease caused by Colletotrichum dracaenophilum: severe wilted and dead lucky bamboo plants showing acervuli on discoloured areas (A). Magnified portion showing acervuli on stem tissue (B).
Table 2.
Morphological characters of Colletotrichum dracaenophilum isolated from lucky bamboo plants.
| Character | Colletotrichum dracaenophilum |
|---|---|
| Colony color | Pale |
| Conidia shape | Hyaline, unicellular, cylindrical to ovoid, straight or slightly curved, guttulate |
| Spore mass color | Pale pink |
| Spore size (μm) | 23–33 × 6.6–9.9 μm (28 × 8.25 μm) |
| Acervuli size (μm) | 180–295 × 3.75–6.25 μm |
| Acervuli on host | Appear |
| Acervuli on media | Appear |
Fig. 2.
Colletotrichum dracaenophilum: Pale aerial mycelium of the fungus growth during seven days at 28 ± 2 °C (A). Conidia produced on media (B). Acervuli with setae produced on lucky bamboo stems (C).
Pathogenicity tests
Pathogenicity on lucky bamboo plants revealed that the fungus C. dracaenophilum caused 100% infection on the inoculated stems of lucky bamboo plants. Two weeks after inoculation, pale green lesions began developing on all inoculated plants, and the fungus was successfully re-isolated. No symptoms were found around the control wounds with pure agar plugs. Anthracnose of lucky bamboo caused by C. dracaenophilum was characterized by the development of small black acervuli on senescent and dead plants Fig. 3. The pathogen infected stems segments and colonized vascular tissues, causing rot of the stem tissue Fig. 4. It is assumed that the maceration of stem and vascular tissues prevents water and nutrient transportation, causing the leaves to wilt and finally the whole plant to die. Lucky bamboo stems injected with C. dracaenophilum conidia showed necrotic, pale green and yellowish lesions around the injection site. The obtained data are consistent with the results obtained recently by Boven et al. [4], Komaki et al. [5] and Sharma et al. [6], who also reported that C. dracaenophilum infected the stems of potted D. sanderiana plants.
Fig. 3.
Artificial inoculation with Colletotrichum dracaenophilum on whole lucky bamboo plants: Dead plants 60 days after inoculation and showing acervuli on stem (A) as compared with healthy plant (B).
Fig. 4.
Stem rot of lucky bamboo segments artificially inoculated with Colletotrichum dracaenophilum conidia after 0, 5 and 15 days of inoculation.
Effect of temperature on the growth of C. dracaenophilum
The effects of different temperatures and incubation periods on the growth of C. dracaenophilum are presented in Table 3. Temperature had a significant effect on the growth of C. dracaenophilum. At both low (5 °C) and high (40 °C) temperatures, fungal growth was completely inhibited during all tested incubation periods. The optimum temperature for C. dracaenophilum growth ranged from 25 to 30 °C, maintained for 8 days. The minimum temperature and maximum average temperature for fungal growth were 10 and 30 °C, respectively. The growth rate was increased by increasing the incubation period from 4 to 14 days at temperatures ranging from 10 to 35 °C.
Table 3.
Effect of temperature on the radial growth (mm) of C. dracaenophilum on PDA medium.
| Incubation period (days) | Radial growth (mm) of C. dracaenophilum | |||||||
|---|---|---|---|---|---|---|---|---|
| Temperature degrees (°C) | ||||||||
| 5 °C | 10 °C | 15 °C | 20 °C | 25 °C | 30 °C | 35 °C | 40 °C | |
| 4 | 0.0 | 00.0 | 11.5 | 21.3 | 41.5 | 50.3 | 16.3 | 0.0 |
| 8 | 0.0 | 08.8 | 23.3 | 32.5 | 90.0 | 90.0 | 34.0 | 0.0 |
| 10 | 0.0 | 11.8 | 33.8 | 48.3 | 90.0 | 90.0 | 47.8 | 0.0 |
| 14 | 0.0 | 20.5 | 45.0 | 77.5 | 90.0 | 90.0 | 55.3 | 0.0 |
| Temperature (T) |
Incubation period (I) |
T × I |
||||||
| L.S.D.0.05 | 2.19 | 2.05 | 2.16 | |||||
Values are mean of four replications for each (T × I) combination for example (5 °C, 4 days).
Effect of different fungicides on the growth of C. dracaenophilum
The effects of different fungicides under different concentrations on the growth of C. dracaenophilum are presented in Fig. 5. Fungicides had a significant effect on the growth of C. dracaenophilum. Among them, Kemazed 50% WP had a strong inhibition effect as no fungal growth was observed over 100 ppm. It was followed significantly by Rizolex T 50% WP, where no fungal growth was observed over 300 ppm. Dithane M-45 and Tridex 80% over 400 ppm completely inhibited the fungal growth. Other fungicides showed moderate inhibitory effect only at 600 ppm. It is also clear that the increase in fungicide concentration had an obvious decrease in the linear growth of C. dracaenophilum.
Fig. 5.
Effect of commercial fungicides on the radial growth (mm) of C. dracaenophilum on PDA medium.
Effect of different bioagents on the growth of C. dracaenophilum
The antagonistic potential of the tested bioagents against the growth of C. dracaenophilum is shown in Table 4, Figs. 6 and 7. Data indicate that most of bioagents had significant antagonistic activity against the growth of C. dracaenophilum. Among bacterial bioagents, B. subtilis and B. pumilus caused the highest growth reduction of C. dracaenophilum. It was followed by P. fluorescens, while B. megaterium showed no antagonistic activity. Among fungal bioagents, T. harzianum, T. viride and T. koningii significantly caused the highest growth reduction of C. dracaenophilum. It was followed by T. virens. Antibiosis and competition for space and nutrients are generally the mode of antagonism observed for Bacillus and Pseudomonas species [21], [22]. Trichoderma strains exert biocontrol against fungal phytopathogens either indirectly, by competing for nutrients and space or directly, by mechanisms such as antibiosis, and mycoparasitism [23].
Table 4.
Effect of some biocontrol agents on C. dracaenophilum grown on PDA medium.
| Biocontrol agent | Radial growth (mm) and reduction (%) of C. dracaenophilum |
|
|---|---|---|
| Radial growth (mm) | Reduction (%) | |
| T. harzianum | 29.3a | 67.4 |
| T. viride | 29.5a | 67.2 |
| T. koningii | 30.8a | 65.8 |
| T. virens | 35.8a | 60.2 |
| B. subtilis | 37.5a | 58.3 |
| B. pumilus | 38.3a | 57.4 |
| P. fluorescens | 66.5a | 26.1 |
| B. megaterium | 90.0b | 00.0 |
| Control | 90.0 | – |
| L.S.D.0.05 | 2.8 | |
Values are mean of four replications for each biocontrol agent as well as the control.
Growth reduction (%) = [(C − T)/C] × 100, Where: C = Average radial growth of C. dracaenophilum in control and T = Average radial growth of C. dracaenophilum in biocontrol agent treatment.
Significantly different from the respective control at P < 0.05.
Not significantly different from the respective control at P < 0.05.
Fig. 6.
Bacterial antagonistic effect on C. dracaenophilum growth. (A) C. dracaenophilum in the presence of P. fluorescens, (B) C. dracaenophilum in the presence of B. pumilus, (D) C. dracaenophilum in the presence of B. megaterium and (E) C. dracaenophilum in the presence of B. subtilis.
Fig. 7.
Fungal antagonistic effect on C. dracaenophilum growth. (A) C. dracaenophilum in the presence of T. harzianum, (B) C. dracaenophilum in the presence of T. viride, (D) C. dracaenophilum in the presence of T. koningii and (E) C. dracaenophilum in the presence of T. virens.
Conclusions
The occurrence of anthracnose symptoms caused by C. dracaenophilum was observed for the first time on D. sanderiana in Egypt. The fungicide Kemazed 50% WP and different biocontrol agents viz., T. harzianum, T. viride, B. subtilis and B. pumilus, restricted the growth of C. dracaenophilum in agar plates.
Conflict of Interest
The authors declared that there is no conflict of interest.
Compliance with Ethics Requirements
This article does not contain any studies with human or animal subjects.
Footnotes
Peer review under responsibility of Cairo University.
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