Anthracnose of Lucky Bamboo Dracaena Sanderiana Caused by the Fungus Colletotrichum Dracaenophilum in Egypt

Journal of Advanced Research (2016) 7, 327–335

Cairo University Journal of Advanced Research

ORIGINAL ARTICLE Anthracnose of lucky bamboo Dracaena sanderiana caused by the fungus Colletotrichum dracaenophilum in Egypt

Ahmed A. Morsy, Ibrahim E. Elshahawy *

Plant Pathology Department, Agricultural and Biological Research Division, National Research Centre, Giza, Egypt

GRAPHICAL ABSTRACT

ARTICLE INFO ABSTRACT

Article history: Dracaena sanderiana, of the family Liliaceae, is among the ornamental plants most frequently Received 4 November 2015 imported into Egypt. Typical anthracnose symptoms were observed on the stems of imported Received in revised form 21 January D. sanderiana samples. The pathogen was isolated, demonstrated to be pathogenic based on 2016 Koch’s rule and identiﬁed as Colletotrichum dracaenophilum. The optimum temperature for Accepted 22 January 2016 its growth ranges from 25 to 30 °C, maintained for 8 days. Kemazed 50% wettable powder Available online 16 February 2016 (WP) was the most effective fungicide against the pathogen, as no fungal growth was observed

* Corresponding author. Tel.: +20 1279180670; fax: +20 57 2403868. E-mail address: [email protected] (I.E. Elshahawy). Peer review under responsibility of Cairo University.

Production and hosting by Elsevier http://dx.doi.org/10.1016/j.jare.2016.01.002 2090-1232 Ó 2016 Production and hosting by Elsevier B.V. on behalf of Cairo University. 328 A.A. Morsy and I.E. Elshahawy

over 100 ppm. The biocontrol agents Trichoderma harzianum and Trichoderma viride followed Keywords: by Bacillus subtilis and Bacillus pumilus caused the highest reduction in fungal growth. To Anthracnose the best of our knowledge, this report describes the ﬁrst time that this pathogen was observed Dracaena sanderiana on D. sanderiana in Egypt. Colletotrichum dracaenophilum Ó 2016 Production and hosting by Elsevier B.V. on behalf of Cairo University. Lucky bamboo

Introduction Pathogenicity tests

Lucky bamboo (Dracaena sanderiana hort. ex Mast.) is among Pathogenicity was confirmed by fulfilling Koch’s postulates on the ornamental plants most frequently imported into Egypt. rooted cuttings of lucky bamboo plants as well as detached This bamboo is also known as Dracaena braunii [1]. Although stem segments, according to Bobev et al. [4]. Twenty cuttings the word bamboo occurs in several of its common names, D. of lucky bamboo plants were surface disinfected with 1.5% sanderiana is actually of an entirely different taxonomic order sodium hypochlorite (NaOCl) for 5 min, followed by several from true bamboos. In Egypt, lucky bamboo is the most popular rinses with sterile distilled water before being sown in five glass indoor plant and is frequently imported and resold in attractive bottles containing 500 mL sterile water. Thirty days later, these pots. Colletotrichum spp. is an imperfect fungus belonging to the bamboo plants were divided into two sets. The stems of the Melanconiales. Members of the genus Colletotrichum cause dis- first set were wounded (ten wounds per plant) using a sterile eases on a number of host plants. These diseases, often referred needle at 4 cm intervals. The stems of 5 plants of the first set to as anthracnose, include strawberry black spot and key lime were inoculated by inserting small mycelial plugs from 10- anthracnose (caused by Colletotrichum acutatum), tomato fruit day-old potato dextrose agar (PDA) cultures of C. dra- anthracnose (caused by Colletotrichum coccodes), red sorghum caenophilum into wounds, which were subsequently covered stalk rot (caused by Colletotrichum graminicola), coffee berry with Parafilm strips. Pure agar plugs were used to inoculate disease (caused by Colletotrichum kawahae), bean anthracnose the wounded stems of the control plants (5 plants). Both inoc- (caused by Colletotrichum lindemuthianum) and many others ulated and control plants were kept at 28 ± 2 °C. Anthracnose [2]. Additional species of Colletotrichum with conidia greater symptoms were observed visually for sixty days after inocula- than 20 lm have been encountered on living plants of D. sande- tion. The stems of the second set (5 plants) were injected with À riana (lucky bamboo) from China [3]. In Bulgaria and Iran, 0.5 mL plant 1 of C. dracaenophilum conidial suspension À Bobev et al. [4] and Komaki et al. [5], respectively, provided (2 Â 106 conidia mL 1) using a sterilized syringe [9]. The the first reports that Colletotrichum dracaenophilum infects the injected and un-injected (5 plants) stems of lucky bamboo stems of potted D. sanderiana plants, causing anthracnose dis- plants were covered with plastic polyethylene bags for 24 h ease. In the United States, Sharma et al. [6] isolated, character- to provide humid conditions. Anthracnose symptoms were ized and tested fungicide treatments to control Colletotrichum observed visually. In addition, the stems of apparently healthy spp. causing anthracnose on lucky bamboo, D. sanderiana. They lucky bamboo plants were cut longitudinally and horizontally also reported that C. dracaenophilum caused the most severe dis- into 1–3 cm segments. These segments were inoculated with ease on lucky bamboo, whereas one isolate of the Colletotrichum one drop of C. dracaenophilum conidial suspension (2 Â 106 À gloeosporioides species complex was less pathogenic to all Dra- conidia mL 1) after surface disinfection. Five stem segments caena spp. and varieties tested. In Egypt, during March 2015, from each type of longitudinal and horizontal pieces were used anthracnose symptoms were recorded on D. sanderiana plants. as replicates, and the experiment was replicated twice. The rot Therefore, the objectives of this work were (i) to describe the of the detached stem segments was observed visually. symptoms of lucky bamboo anthracnose, (ii) to isolate, identify and test the pathogenicity of the causal agent, (iii) to determine Effect of temperature on the growth of C. dracaenophilum the effect of temperature on the growth of the causal pathogen and (iv) to evaluate the effect of certain fungicides and biocon- Fresh potato dextrose agar (PDA) plates were inoculated with trol agents on the growth of the pathogen. a 5 mm mycelial disk cut with a sterile cork borer from the margin of a 10-days-old colony of C. dracaenophilum. Plates Material and methods were incubated in an incubator at 5, 10, 15, 20, 25, 30, 35 and 40 °C. The radial growth of C. dracaenophilum was mea- Isolation and identification of the causal pathogen sured in two perpendicular directions at 4, 8, 10 and 14 days after inoculation. Four Petri plates were used as replicates In March 2015, disease problems on the stems of imported for each combination of temperature and incubation period. (Netherlands) indoor lucky bamboo plants (D. sanderiana) were observed. The symptoms were observed during several months Inhibitor effect of fungicides on the growth of C. dracaenophilum after the consumer purchased them from the retail stores located in Giza governorate. More than 50 diseased samples with typi- The inhibition effects of ten different fungicides under different cal anthracnose symptoms were collected to isolate the patho- concentrations viz., 0, 25, 50, 100, 200, 300, 400, 500 and gen based on Koch’s rule [7]. The obtained fungal colonies 600 ppm against the pathogen were determined. The systemic were identified according to the Colletotrichum description fungicides were dimethomorph 6% + copper oxychloride reported by Sutton [8] and according to Farr et al. [3]. 40% (Acrobat Copper 46%), carbendazim (Kemazed 50% Anthracnose of lucky bamboo Dracaena sanderiana in Egypt 329

Table 1 Analysis of variance for RCBD. SOV Table 3 Table 4 df P-values df P-values Replication (r À 1) = 3 0.48 (r À 1) = 3 <0.01 A (temperature T or fungicide F) A À 1 = 7 <0.01 A À 1 = 9 <0.01 B (incubation period I or concentration C) B À 1 = 3 <0.01 B À 1 = 7 <0.01 AB (A À 1) (B À 1) = 21 <0.01 (A À 1) (B À 1) = 56 <0.01 Error (AB À 1) (r À 1) = 93 (AB À 1) (r À 1) = 237 Total (ABr À 1) = 127 (ABr À 1) = 314

WP), flutolanil (Moncut 25% WP), mancozeb (Tridex 80%), C = Average growth of C. dracaenophilum in control and metalaxyl M + mancozeb (Ridomil Glod 68%), and T = Average growth of C. dracaenophilum in biocontrol thiophanate-methyl (Topsin-M 70% wettable granul (WG) agent treatment. and the protective fungicides were Mancozeb 80% (Dithane M-45), pencycuron (Monceren 25% WP) and thiram Statistical analysis + tolclofos-methyl (Rizolex T 50% WP). The Metalaxyl 8% WP + Mancozeb 64% (Tasoline) is systemic and protective Statistical analysis for a randomized complete block design fungicide. The inhibition effect was tested using the poisoned (RCBD) with two factors and interaction terms was performed food technique described by Uribe and Loria [10]. Four Petri for all experiments according to Gomez and Gomez [14]. Least plates were used as replicates for each treatment as well as significant difference (LSD) values were calculated to test the untreated control. The average radial growth of C. dra- significance of differences between means according to Steel caenophilum was measured in two perpendicular directions et al. [15] (Table 1). when C. dracaenophilum reached full growth in the control plate. Results and discussion Inhibitor effect of biocontrol agents on the growth of C. Symptomatology and the causal pathogen dracaenophilum

The primary symptoms of lucky bamboo anthracnose were Fungal and bacterial biocontrol agents viz., Trichoderma pale green yellowish lesions that appeared on the stems. harzianum, Trichoderma viride, Trichoderma virens, Tricho- These symptoms extended to the upper and lower intern- derma koningii, Pseudomonas fluorescens, Bacillus subtilis, odes, which became yellow. The hard tissues turned soft, Bacillus megaterium and Bacillus pumilus were obtained from the plant showed wilt symptoms, and the entire stem was the Plant Pathology Department, National Research Centre covered with numerous black globose ellipsoid acervuli with (NRC). The inhibitor effect of the fungal biocontrol agents sparse, black setae Fig. 1. The pathogen was isolated and against the growth of C. dracaenophilum was studied using identified as C. dracaenophilum D. F. Farr & M. E. Polm, the method described by Bell et al. [11]. Petri plate contain- according to the classification of Sutton [8] and Farr et al. ing PDA medium was inoculated on one side with a 5 mm [3]. C. dracaenophilum has been reported in many regions, mycelial disk from a 7-day-old culture of the test fungus. such as Cyprus [16], China and Kenya [18], Bulgaria, [4], The opposite side was inoculated with a disc of C. dra- Iran [5], and the United States [17], including south Florida caenophilum and the plates were incubated at 28 ± 1 °C. and retail stores in north Florida [6]. This work is the first Plates inoculated with a disc of C. dracaenophilum by itself report of this fungal species in Egypt. The morphological were used as a control. Four replicate plates were made characteristics of the pathogen are shown in Table 2 and for each test fungus as well as the control. Colony radius Fig. 2. Colonies of the fungus grown on PDA were domi- of C. dracaenophilum was recorded when the control plates nated by pale aerial mycelium. The acervuli on dying stems, reached full growth. The inhibitory effect of the bacterial numerous in the discolored areas, were arranged concentri- biocontrol agents was studied using the method described cally on the stem, forming tiny black spots, 264–382.8 lm by Estrella et al. [12]. Petri plate containing PDA medium in the longest dimension, with septate setae, sparse, scattered was inoculated (by streaking) on one side with one loopful in the hymenium, black, 180–295 Â 3.75–6.25 lm, straight to from a 48-h-old culture of the test bacterium. The opposite slightly curved, becoming narrow and hyaline at the rounded side was inoculated with a disc of C. dracaenophilum and the apex, 4–11 septet. Acervuli were also produced on PDA plates were incubated at 28 ± 1 °C. Plates inoculated with a medium. On the plant and in culture, the conidia were hya- disc of C. dracaenophilum by itself were used as a control. line broadly clavate to cylindrical, occasionally slightly Four replicate plates were made for each test bacterium as curved, and measured 23–33 Â 6.6–9.9 lm (28 long Â 8.25 well as the control. Colony radius of C. dracaenophilum width lm). The pathogen can be expected anywhere in the was recorded when the control plates reached full growth. world where infected lucky bamboo cuttings are imported The reduction in the growth of C. dracaenophilum was calcu- from China [6]. Even in the Netherlands, the cuttings actu- lated using the formula suggested by Pandy et al. [13] as fol- ally come from China. Sinclair [19] and Verhoeff [20] stated lows: Growth reduction (%) = [(C À T)/C] Â 100, Where: 330 A.A. Morsy and I.E. Elshahawy

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 (lm) 23–33 Â 6.6–9.9 lm (28 Â 8.25 lm) Acervuli size (lm) 180–295 Â 3.75–6.25 lm Acervuli on host Appear Acervuli on media Appear

that even in the absence of visible symptoms, Colletotrichum black acervuli on senescent and dead plants Fig. 3. The patho- spp. may persist on plants as microscopic latent infections gen infected stems segments and colonized vascular tissues, consisting of appressoria with limited development of infec- causing rot of the stem tissue Fig. 4. It is assumed that the tive hyphae. Sharma et al. [6] made the same observation maceration of stem and vascular tissues prevents water and and reported that lucky bamboo introduced from China nutrient transportation, causing the leaves to wilt and ﬁnally might carry C. dracaenophilum, which can induce anthrac- the whole plant to die. Lucky bamboo stems injected with C. nose symptoms several months after arrival in the United dracaenophilum conidia showed necrotic, pale green and yel- States. It is not known which environmental factors might lowish lesions around the injection site. The obtained data trigger the appearance of symptoms. However, Sharma are consistent with the results obtained recently by Boven et al. [6] mentioned that anthracnose lesions appeared on et al. [4], Komaki et al. [5] and Sharma et al. [6], who also non-inoculated stalks of D. sanderiana plants when the irri- reported that C. dracaenophilum infected the stems of potted gation intervals were lengthened. Thus, water stress may D. sanderiana plants. trigger symptoms. Effect of temperature on the growth of C. dracaenophilum Pathogenicity tests The effects of different temperatures and incubation periods Pathogenicity on lucky bamboo plants revealed that the fun- on the growth of C. dracaenophilum are presented in Table 3. gus C. dracaenophilum caused 100% infection on the inocu- Temperature had a signiﬁcant effect on the growth of C. dra- lated stems of lucky bamboo plants. Two weeks after caenophilum. At both low (5 °C) and high (40 °C) tempera- inoculation, pale green lesions began developing on all inocu- tures, fungal growth was completely inhibited during all lated plants, and the fungus was successfully re-isolated. No tested incubation periods. The optimum temperature for C. symptoms were found around the control wounds with pure dracaenophilum growth ranged from 25 to 30 °C, maintained agar plugs. Anthracnose of lucky bamboo caused by C. dra- for 8 days. The minimum temperature and maximum average caenophilum was characterized by the development of small temperature for fungal growth were 10 and 30 °C, respectively. Anthracnose of lucky bamboo Dracaena sanderiana in Egypt 331

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).

The growth rate was increased by increasing the incubation Effect of different bioagents on the growth of C. dracaenophilum period from 4 to 14 days at temperatures ranging from 10 to ° 35 C. The antagonistic potential of the tested bioagents against the growth of C. dracaenophilum is shown in Table 4, Effect of different fungicides on the growth of C. Figs. 6 and 7. Data indicate that most of bioagents had signif- dracaenophilum icant antagonistic activity against the growth of C. dracaenophilum. Among bacterial bioagents, B. subtilis and B. The effects of different fungicides under different concentra- pumilus caused the highest growth reduction of C. dra- tions on the growth of C. dracaenophilum are presented in caenophilum. It was followed by P. fluorescens, while B. mega- Fig. 5. Fungicides had a significant effect on the growth of terium showed no antagonistic activity. Among fungal C. dracaenophilum. Among them, Kemazed 50% WP had a bioagents, T. harzianum, T. viride and T. koningii significantly strong inhibition effect as no fungal growth was observed over caused the highest growth reduction of C. dracaenophilum.It 100 ppm. It was followed significantly by Rizolex T 50% WP, was followed by T. virens. Antibiosis and competition for where no fungal growth was observed over 300 ppm. Dithane space and nutrients are generally the mode of antagonism M-45 and Tridex 80% over 400 ppm completely inhibited the observed for Bacillus and Pseudomonas species [21,22]. Tricho- fungal growth. Other fungicides showed moderate inhibitory derma strains exert biocontrol against fungal phytopathogens effect only at 600 ppm. It is also clear that the increase in either indirectly, by competing for nutrients and space or fungicide concentration had an obvious decrease in the linear directly, by mechanisms such as antibiosis, and mycopara- growth of C. dracaenophilum. sitism [23]. 332 A.A. Morsy and I.E. Elshahawy

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).

0 Days 5 Days 15 Days

Fig. 4 Stem rot of lucky bamboo segments artiﬁcially inoculated with Colletotrichum dracaenophilum conidia after 0, 5 and 15 days of inoculation. Anthracnose of lucky bamboo Dracaena sanderiana in Egypt 333

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 °C10°C15°C20°C25°C30°C35°C40°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 Â I

L.S.D.0.05 2.19 2.05 2.16 Values are mean of four replications for each (T Â I) combination for example (5 °C, 4 days).

Fig. 5 Effect of commercial fungicides on the radial growth (mm) of C. dracaenophilum on PDA medium.

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 À T)/C] Â 100, Where: C = Average radial growth of C. dracaenophilum in control and T = Average radial growth of C. dracaenophilum in biocontrol agent treatment. a Signiﬁcantly different from the respective control at P < 0.05. b Not signiﬁcantly different from the respective control at P < 0.05. 334 A.A. Morsy and I.E. Elshahawy

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. Anthracnose of lucky bamboo Dracaena sanderiana in Egypt 335

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