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Chemical and Biological Control of Dracaena marginata Leaf Spots in Northern Egypt.

Byline: A. Hilal, Eman El-Argawy, A. El. Korany and Taghreed Fekry

Abstract

Six common fungicides, Dithane M45, Kocide 2000, Ridomil Gold Plus, Score, Equation Pro and Kemazed were tested for controlling the draceana leaf spot fungi. All the tested fungicides significantly decreased the in vitro growth of the tested dracaena leaf spot fungi. Percentage of inhibition on colony diameter ranged between 49.64% with Dithane M45 to 82.18% with Score which was followed by Kemazed (69.15%). However, inhibition of spore germination was the highest (72.8%) by Dithane M45, while inhibition of germ tube reached 89.91% with Kemazed. Meanwhile, in the in vivo tests, treatment with Kemazed decreased disease incidence of leaf spots on potted plants to 16.67% compared to 100% for the untreated inoculated control. On the other hand, the bacterial and fungal biocontrol agents tested proved to be effective against the dracaena leaf spot fungi.

The in vitro assays conducted showed that the bacterial biocontrol agents namely, Stenotrophomonas maltophilia, Pseudomonas ultimum, P. fluorescens, P. putida, P. aeruginosa, Bacillus subtilis, B. megaterium, significantly decreased colony growth diameter (76.84-45.69%) of dracaena leaf spot fungi as well as spore germination (48.80-23.20%) and length of germ tube (88.77-65.65%) of the germinated spores compared to untreated control. Treatment with S. maltophilia showed the most consistent highest inhibition effect, followed by P. ultimum, B. subtilis, and B. megaterium, respectively. The in vivo tests of the most effective bacterial bioagents supported the in vitro results.

Treatment with S. maltophilia showed the highest effect as it decreased disease incidence and disease index to 29.33% and 7.6%, respectively in comparison with 100% and 83.6% for the untreated inoculated control in the potted dracaena plants (cv. Bicolor). Meanwhile, the in vitro tests of the three Trichoderma spp., i.e. T. harzianum, T. hamatum and T. album showed significant inhibition effect on colony growth diameter of the tested dracaena leaf spot fungi in which T. harzianum showed the highest inhibition effect (75.02%), while T. album was most effective to inhibit spore germination (48%) and germ tube (90.21%) of the germinated spores. The in vivo tests of the fungal biocontrol agents supported the in vitro results. T. album (Bio-Zeid) showed the highest effect and decreased disease incidence and disease index on potted plants to 19.99% and 4.39%, respectively compared with 100% and 92.8% for the untreated inoculated control.

Keywords: Dracaena; Leaf spots; Chemical; Biological control

Introduction

Dracaena is one of the most popular and widespread ornamental plants all over the world including Egypt. It is becoming an economically important part of the horticultural industry. Unfortunately, dracaena plants are attacked by several pathogens causing leaf spot diseases. Fungal leaf spots have become increasingly serious and such diseases can lead to heavy defoliation that causes serious losses in the nursery production (Hilal et al., 2000).

Chemical protection is a primary strategy in the control of plant diseases. Using fungicides is the most common way to protect plants from diseases and this is a widely accepted in the absence of resistant source. However, application of fungicides is not always efficient. In addition, the hazards and the environmental pollution caused by agrochemicals used for plant diseases control is a worldwide problem. Bio and natural disease control agents might have less risk to the other living beings and also to the environment. However, little literature is available concerning the use of bacterial biocontrol agents against leaf spot diseases of dracaena.

Trichoderma is a fast growing fungus, strong spore producer, source of cell wall degrading enzymes and an important antibiotic producer. Application of Trichoderma spp. as biocontrol agents can also, bring substantial changes in plant metabolism to promote plant growth, increase nutrient availability, improve crop production and enhance disease resistance (Harman et al., 2004).

In Egypt, little information exists on the chemical and biological control of dracaena fungal leaf spots in Egypt.

Therefore, the present study was conducted to determine the efficacy of certain chemical fungicides in vitro and in vivo for controlling dracaena leaf spot fungi, and the efficacy of certain bacterial and fungal biocontrol agents against dracaena leaf spot fungi.

Materials and Methods

Chemical Control

In vitro Effect of Fungicides on Colony Growth of Dracaena Leaf Spot Fungi

The tested fungi: Five of the most pathogenic leaf spot fungi of dracaena namely Botryodiplodia theobromae, Fusarium graminearum, F. proliferatum, F. verticillioides and Gibellulopsis nigrescens were obtained from fungal collection established by the authors in Faculty of Agriculture, Damenhour University.

The tested fungicides: Six fungicides commonly used to control leaf spots were tested in vitro in the present study. Fungicides were used as diluted solutions or suspension in water at their recommended doses. Fungicides name (commercial and/or common), formulation, rate of application and manufacture source are listed in Table (1).

The recommended rate of each fungicide was added to sterilized PDA medium before solidification to obtain the desired concentrations. Precisely 20 mL of un amended (control) or amended PDA for each treatment was poured into each of five Petri dishes. After solidification of the medium, each dish was inoculated centrally with a mycelial disc (5 mm diameter) taken from the margins of actively growing mycelium from PDA cultures of each tested fungal isolate. Plates were incubated at 26 +- 2degC. The average of colony diameter was measured when the untreated control mycelia had just covered the whole plate.

Percentage of inhibition (efficacy): The percentage of inhibition (efficacy) for each fungicide in Petri dishes was calculated according to the following formula (Amer, 1995):

Inhibition % (Efficacy) = (A- B/ A) x 100

Where: A = Diameter of fungal colony in un-amended medium (control).

B = Diameter of fungal colony in amended medium.

Effect of Fungicides on Spore Germination and Germ Tube Length

Spore suspension of each tested fungus was prepared by pouring 20 mL of sterile distilled water on fungal culture in each Petri dish with gentle rubbing of culture surface to harvest spores. Then fungicides were added to spore suspension and fungicide concentrations were adjusted. Three drops of 0.1 mL of each tested fungicide containing spore suspension were put on clean glass of germination slide, other slides with drops of sterilized distilled water were used as control and they were incubated at 26 +- 2degC, for 6 h. Then slides were examined under light microscope to count the germinated and ungerminated spores with a haemocytometer. Length of germ tube of the germinated spores was measured, using ocular micrometer which was calibrated by a micrometer slide (Shama and Massoud, 2001). The values were converted to microns using the conversion multiplier established when the microscope was calibrated. The length of at least 10 germinated spores was measured and recorded.

In Vivo Effect of Fungicides on Leaf Spot Incidence

The in vitro most effective fungicides for controlling dracaena leaf spot fungi were tested in vivo under greenhouse conditions on Dracaena marginata (cv. Bicolor).

Fungi and Inocula

The five most pathogenic dracaena leaf spots fungi previously tested in vitro were used for the in vivo test. Pure cultures of the tested fungi were grown separately on 100 mL autoclaved potato dextrose liquid (PDL) in 250 mL conical flasks and incubated at 26+-2degC for 15-20 days to obtain sufficient mycelial growth. The fungal mycelium of each pathogen was collected and blended with 100 mL sterilized distilled water in a blender for one minute. Concentration of the fungal propagate suspensions (spores or mycelia fragments of the sterile fungus) was determined (El-Abd, 2002). The spore concentration was adjusted using a haemocytometer and concentration of inocula were 18x106 spores/mL for Botryodiplodia theobromae, 09x106 for F. graminearum, 05x106 for F. proliferatum, 07x106 for F. verticillioides and 04x106 for Gibellulopsis nigrescens according to pre-experiment conducted by the authors.

Healthy Dracaena Plants

The used dracaena plants (cv. Bicolor) were obtained from private farm of Bissar "Violette" nurseries at Ras El-Soda, Alexandria, where they were imported from Holland. They were used as seedlings of 11 months old.

Soil

Soil used for filling the pots in pathogenicity tests consisted of a mixture of sand, peat moss, loam and perlite at volumes rates of 4:3:2:1, respectively. Sand and loam were sterilized by autoclaving at 121degC for 30-60 mins during a period of sequenced three days. Pots of 25 cm in diameter were used through the greenhouse trials for planting (1 plant/pot). Pots were sterilized by immersing them in 5% formalin solution for 15 min and left to dry and to get rid of formalin odor.

The Detached Leaves Assay

Apparently healthy leaves (No. 6-8) of dracaena (cv. Bicolor) were detached, sterilized with 70% ethanol and placed in separate plastic bags with moistened tissue paper to create humid conditions. Then the leaves were inoculated with the appropriate concentration of each fungal spore suspension, five replicates for each treatment. The leaves were then incubated at room temperature 23 +- 5degC and daily examined over two weeks of incubation for full development of lesions. Then disease incidence and disease index were assessed (Jackson et al., 2008; Boydom et al., 2013).

Potted Plants Assay

This experiment was conducted under greenhouse conditions at 23-26degC during the day and at 17-19degC at night using dracaena plants (cv. Bicolor) of 11 month old seedlings. Fungicides were sprayed, 24 h after fungal inoculation (curative treatment). Five pots (replicates), each containing one plant were used for each treatment. Untreated plants (control) were sprayed with distilled water. All plants were covered with polyethylene bag for 48 h to maintain high relative humidity. Plants were kept in greenhouse under daily observation for 14 days. Measurements of disease incidence and index (severity) were carried out.

Disease Incidence

Disease incidence was determined as the percentage of spotted plants (or leaves) relative to the total number of the examined ones according to Morang et al. (2012) as follows:

(Equation)

Disease Index

Each plant (or plant leaf) showing symptoms of the leaf spots was examined with care and a rating was given according to Abd El-Zaher et al. (2005), applying the following numerical ratings:

0 = No disease symptoms (apparently immune).

1 = Few scattered lesions covering about 1-25% of the leaf (mild infection).

2 = Spots covering about 25-50% of the leaf (moderate infection).

3 = Spots coalescing and covering about 50-75% of the leaf (high infection).

4 = Infection with coalescing lesion covering more than 75% of the leaf (very severe infection).

5 = 100% of leaf covered with spots (extremely severe infection).

Disease index was estimated according to the equation suggested by Abd El-Zaher et al., 2005).

(Equation)

Where: S (nr) = Total [number of plants (or leaves) under scale degree x scale degree].

5N =Degree of freedom of scale degrees (5) x total number of plants (or leaves) examined.

Biological Control

The tests were conducted using certain bacterial and fungal biocontrol agents (Table 2) in vitro and in vivo to evaluate their efficacy against the most pathogenic fungi of the dracaena leaf spots previously tested under chemical control. The fungal isolates tested were obtained from the authors fungal collection in Plant Pathology Dept. Faculty of Agriculture, Damanhour University. The used biocontrol agents were obtained from Plant Pathology Research Institute, Agric. Res. Center, Giza, Egypt.

Efficacy of Bacterial Biocontrol Agents for Controlling Dracaena Leaf Spot Fungi

In vitro effect on colony growth: The bacterial biocontrol agents were grown in nutrient agar medium in 250 mL flasks containing 50 mL of the medium. Seventy two hours after incubation, 10 mL of physiologic solution was added to each flask with shaking to harvest bacteria and then bacterial concentrations were adjusted. Bacterial suspension was added to warm sterilized PDA medium at the rate of 10%. Approximately 20 mL of non-amended (control) or amended PDA for each treatment were poured into each of five Petri dishes. Each dish was inoculated centrally by a mycelial disk (5 mm diameter) of pathogenic fungi taken from the margins of actively growing mycelium of PDA cultures. Plates were incubated at 26 +- 2degC.

Fungal growth diameters were measured after fungal growth covered completed any one of the untreated plates and percentage of inhibition (efficacy) of bio-agents, as reduction in colony diameter, was calculated as previously mentioned.

In vitro effect on spore germination and germ tube length: The experiment was conducted as previously mentioned under effect of the fungicides on spore germination and germ tube length.

In vivo effect of bacterial biocontrol agents on dracaena leaf spot disease: The most effective bacterial bio-agents, in vitro, were tested under greenhouse conditions for controlling fungal leaf spot on D. marginata. The experiment was conducted as previously mentioned under the in vivo effect of fungicide and the bacterial bio-agents were prepared as mentioned under the in vitro test of bacterial biocontrol agents.

Efficacy of Fungal Biocontrol Agents for Controlling Dracaena Leaf Spot Fungi

In vitro effect on colony growth: Bio-control agent cultures of Trichoderma spp. were prepared from Plant- guard(r) and Bio Zeid(r) by adding one ml of stock solution in water of each to pure PDA medium plates before solidification with shaking. All plates were incubated at 26+-2degC for 5 days.

Two plugs (5-mm diam.) were taken, one from the advancing margins of seven day old cultures of the tested fungi and the other from the biocontrol agents Trichoderma spp. (Table 2). The two plugs were placed at 1.5 cm from the edge of the plate at opposite sides (Mueller et al., 1985).

Plates were then incubated at 26 +- 2degC for 7 days. Control plates were inoculated with plugs of PDA free of the bio-agents. Five replicates were used for each treatment. Diameter of the developed tested fungal colonies was measured and percentage of inhibition (efficacy) was calculated as previously mentioned under the effect of fungicides.

In vitro effect on spore germination and germ tube: The experiment was conducted as previously mentioned under the effect of fungicides on spore germination.

In vivo tests: The most effective Trichoderma biocontrol agents which were tested firstly in vitro on dracaena leaf spot fungi were tested also under greenhouse conditions on D. marginata "cv. Bicolor". The test was conducted as previously mentioned under the in vivo effect of fungicide.

Statistical Analysis

The obtained data were statistically analyzed according to Gomez and Gomez (1984) using SAS (Statistical Analysis System) version 9.2, 2001. Before carrying out analysis of variance (ANOVA), the percentages were transformed to angular transformation according to Steel and Torrie (1984). Means of treatments were compared using the least significant difference test at 5% level of probability.

Results

Chemical Control

In vitro effect of fungicides on colony growth of dracaena leaf spot fungi: The tested fungicides obviously suppressed the colony diameter of all the tested leaf spot fungi (Fig. 1). Score fungicide followed by Kemazed proved to be the most effective. Their means of inhibition of colony diameter were 82.18% and 69.15%, respectively (Table 3). Meantime, Kocide 2000 exhibited 59.70% of inhibition followed by Ridomil Gold, Equation Pro and Dithane M45 where 57.69%, 55.24% and 49.64% of inhibition were recorded, respectively (Table 3).

In vitro effect of fungicides on spore germination and germ tube of dracaena leaf spot fungi: All fungicides tested significantly inhibited spore germination of the tested dracaena leaf spot fungi (Table 4). Overall means of percentage of inhibition ranged between 55.6% with Kemazed to 72.8-72.4% with each of Dithane M45 and Kocide 2000 (Table 4). Meanwhile, the fungicides significantly inhibited growth of germ tube of the germinated spores of the different fungi, where Kemazed and Score exhibited the highest inhibition effect being 89.91% and 88.25%, respectively. This was followed by Ridomil Gold, Equation Pro, Kocide 2000 and Dithane M45 where inhibitions were 84.26, 81.32, 77.45 and 74.89%, respectively (Table 4).

In vivo Effect of Fungicide on Dracaena Leaf Spot Disease

Effect on detached leaves: Data presented in Table (5) and illustrated in Fig. (2) showed that foliar application with the tested fungicide as curative treatment significantly inhibited disease incidence and disease index of dracaena leaf spots caused by the inoculation with five pathogenic leaf spot fungi of dracaena. Kemazed was the most effective and decreased mean percentage of infection to 31.11% compared to 100 % for the infected untreated control. This was followed by Score, where percentage of infection decreased to 48.89%, while Equation Pro exhibited the lowest effect. Similar trend was revealed for the disease index (Table 5; Fig. 2). However, Kemazed exhibited more pronounced effect as it decreased the leaf spot disease index 7.56% compared to 92.8% for the inoculated untreated control. This was followed by Score with 12.44% disease index while Equation Pro exhibited the lowest effect being 27.15% disease index (Table 5; Fig. 2).

Effect on potted plants: Data in Table (6) showed that spraying Kemazed, which was most effective in the detached leaf assay, to dracaena potted plants (cv. Bicolor) previously inoculated with dracaena leaf spot fungi, significantly decreased mean percentage of infection to 16.67% compared to 100% for the untreated inoculated control. Meantime, the developed mean disease index decreased to 4.13% compared to 83.6% for the untreated inoculated control.

Biological Control

Efficacy of bacterial biocontrol agents - in vitro effect on colony diameter of dracaena leaf spot fungi: Incorporating the biocontrol bacterial bio agents into PDA obviously inhibited colony growth of the tested leaf spot fungi (Fig. 3). Meanwhile, data in Table (7) showed that Stenotrophomonas maltophilia exhibited the highest mean inhibition value (76.84%). This was followed by Pseudomonas ultimum (60.18%) and Bacillus subtilis (60.53%) with no significant variation between the last two bacterial bioagents (Table 7).

Table 1: The tested fungicides, common name, source and rate of application

Commercial name and###Dithane M45(r)###Kocide 2000(r)###Ridomil Gold plus(r)###Score(r)###Equation Pro(r)###Kemazid(r) 50% WP

formulation###80% WP###53.8% WP###42.58% WP###25% E.C###52.5% WG

Recommended rate/100 L###20 g###180 g###200 g###50 ml###40 g###75 g

Common name###Mancozeb###Cu-hydroxide###Copper oxychloride###Difenocon +###Cymoxanil +###Carbendazim

###Famoxadone###Famoxadone

Source (Manufacture)###Egyptian grow###Sentic###Syngenta###Novartis###Syngenta###Kafr EL-Zyat Co. for pesticides (Egypt)

Table 2: List of biocontrol agents and their rate of application used in the present study

Biocontrol Agents###Scientific name (preparation)###Commercial name (Common name)

###Bacillus megaterium; Powder (25*106 cell/g) and used at concentration of 250 g/100 L water###Bio Arc(r)

###B. subtilis; Powder (30 * 106 cell/g) and used at concentration of 4 g/L water###Rhizo-N(r)

###B. cereus (28*106)###isolate

Bacterial Agents###B. thuringiensis (28*106)###isolate

###Stenotrophomonas maltophilia (40*106)###isolate

###Pseudomonas flurocens (33*106)###isolate

###P. putida (37*106)###isolate

###P. ultimum (36*106)###isolate

###P. aeruginosa(36*106)###isolate

Fungal Agents###Trichoderma album; Powder (25 * 106 spores/g) and used at concentration of 250 g/100 L water###Bio Zeid(r)

###T. harzianum; (8 * 107 spores/ml) and used at concentration of 6 g /100 L /water###Plant Guard(r)

###T. hamatum (8 * 106)###Isolate

Table 3: The in vitro colony diameter of five pathogenic dracaena leaf spot fungi on PDA amended with six different fungicides

Leaf spot###fungi Botryodiplodia F. graminearum###F. proliferatum F. verticil-###Gibellulopsis###Means

Fungicides###theobromae###lioides###nigrescens###Colony diameter###Inhibition (efficacy) %

Dithane M45###6.60###2.70###3.76###2.98###6.62###4.53###49.64 F

Kocide 2000###5.58###1.56###4.46###4.28###2.24###3.62###59.73 C

Ridomil Gold###7.20###1.50###3.80###4.52###2.02###3.80###57.69 D

Equation Pro###5.32###2.38###4.20###3.66###4.58###4.03###55.24 E

Kemazed###2.22###1.84###3.50###2.66###3.66###2.78###69.15 B

Score###1.50###1.64###1.72###1.66###1.50###1.60###82.18 A

Control###9.00###9.00###9.00###9.00###9.00###9.00###0.00 G

Table 4: Percentage of spore germination, length of germ tube and inhibition of five pathogenic dracaena leaf spot fungi caused by the tested six fungicides

Fungi###Spore Germination (%)###Germ tube length (um)

Fungicides###B.###F.###F.###F.###G.###Mean###B.###F.###F.###F.###G.###Mean

###theobromae graminearum prolife-###verticil-###nigrescens inhibition theobromae###graminearum prolife- vertici-###nigrescens inhibition

###ratum###lioides###(%)###ratum###llioides###(%)

Dithane###26.00###20.00###26.00###34.00###30.00###72.80A###0.27###0.19###0.25###0.17###0.18###74.89E

M45

Kocide###26.00###24.00###24.00###24.00###40.00###72.40A###0.21###0.18###0.25###0.15###0.16###77.45D

2000

Ridomil###22.00###24.00###32.00###36.00###44.00###68.40B###0.13###0.15###0.16###0.11###0.12###84.26B

Gold

Equation###38.00###30.00###30.00###34.00###30.00###67.60B###0.11###0.14###0.18###0.14###0.26###81.32C

Pro

Kemazed###36.00###38.00###56.00###52.00###40.00###55.60C###0.10###0.10###0.05###0.07###0.14###89.91A

Score###52.00###28.00###50.00###32.00###46.00###58.40C###0.06###0.15###0.08###0.15###0.09###88.25A

Control###100.00###100.00###100.00###100.00###100.00###0.00D###0.75###1.064###0.774###0.938###0.906###0.00F

This was followed by P. aeruginosa, B. megaterium and P. fluorescens, where inhibitions were 57.29%, 56.89% and 55.38%, respectively. Rest of the bacterial bioagents, i.e. B. thuringiensis, P. putida and B. cereus exhibited inhibition <50%.

In vitro effect on spore germination and germ tube: All bacterial bioagents tested significantly decreased spore germination of the tested fungal isolates (Table 8). Percentage of inhibition of spore germination ranged between 48.8% with S. maltophilia to 44.0% with B. subtilis and 36% with P. ultimum. The bioagents i.e., P. putida, B. megaterium, P. aeruginosa, came next with inhibition effect ranged between 29.6% and 28.0%, while P. fluorescens exhibited the lowest effect (23.20%). Bacterial bioagents also significantly decreased germ tube length (um) of the germinated spores of the tested dracaena leaf spot fungi (Table 8). Percentage of inhibition ranged between 88.77% with S. maltophilia to 67.15% with each of B. cereus and B. thuringiensis (Table 8). However, B. subtilis, P. ultimum, B. megaterium, P. aeruginosa, and P. putida showed percentage of inhibition of 84.97%, 80.45%, 73.88%, 73.0% and 72.15%, respectively (Table 8).

In vivo Effect of Bacterial Biocontrol Agents on Dracaena Leaf Spot Disease

Effect on detached leaves: Data presented in Table (9) illustrated in Fig. 4 showed that treatment with the tested bacterial bioagents (curative) to detached dracaena (cv. Bicolor) leaves inoculated with dracaena leaf spots fungi, significantly decreased disease incidence and disease index of leaf spots developed by the inoculated fungi. Meanwhile, S. maltophilia showed the highest effect as decreased percentage of infection to 53.33% compared to 100% for the inoculated untreated control. This was followed by B. megaterium, which decreased percentage of infection to 64.44%. The other bacterial bioagents i.e., P. ultimum and B. subtilis, decreased percentage of infection to 73.33% and 75.55%, respectively compared to 100% for the untreated infected control. Similar trend was revealed for the disease index as S. maltophilia significantly decreased the leaf spot disease index to 16.0% compared to 92.8% for the inoculated untreated control (Table 9 ; Fig. 4).

Table 5: Percentage of infection and disease index of leaf spots developed on dracaena (cv. Bicolor) inoculated with five dracaena leaf spot fungi and sprayed with certain fungicides, using the detached leaves technique

Dracaena leaf spot fungi###Percentage of infection (%)###Disease index (%)

###Infected and treated###Infected###Infected and treated###Infected

###Equation Pro###Kemazed###Score###un treated###Equation Pro###Kemazed###Score###un treated

Botryodiplodia theobromae###88.89###44.44###66.70###100.00###26.67###13.33###17.80###90.00

Fusarium graminearum###88.89###33.33###44.44###100.00###35.56###8.89###13.30###96.00

Fusarium proliferatum###77.78###11.11###44.44###100.00###20.00###2.22###11.10###92.00

Fusarium verticillioides###88.89###44.44###55.56###100.00###33.33###8.89###13.30###94.00

Gibellulopsis nigrescens###77.78###22.22###33.33###100.00###20.20###4.44###6.70###92.00

Mean###84.45 B###31.11 D###48.90 C###100 A###27.15 B###7.56 D###12.44 C###92.80 A

Table 6: Disease incidence and disease index of leaf spots developed on potted dracaena plants (cv. Bicolor) inoculated with dracaena leaf spot fungi and sprayed with kemazed fungicide under greenhouse conditions

Dracaena leaf spot fungi###Percentage of infection (%)###Disease index (%)

###Infected and treated###Infected un treated###Inhibition (%)###Infected and treated###Infected un treated###Inhibition (%)

Botryodiplodia theobromae###23.33###100.00###76.67 E###6.00###82.00###92.68 B

Fusarium graminearum###20.00###100.00###80.00 D###5.33###90.00###94.07 AB

Fusarium proliferatum###10.00###100.00###90.00 A###2.00###80.00###97.50 A

Fusarium verticillioides###16.67###100.00###83.33 C###4.67###84.00###94.44 AB

Gibellulopsis nigrescens###13.33###100.00###86.67 B###2.67###82.00###96.70 A

Mean###16.67 B###100 A###83.33###4.13 B###83.60 A

Table 7: The in vitro colony diameter and percentage of inhibition of five dracaena leaf spot fungi on PDA amended with bacterial biocontrol agents

Leaf spot fungi Bacterial bio Botryodiplodia###Fusarium###Fusarium###Fusarium###Gibellulopsis###Mean

agents###theobromae###graminearum###proliferatum###verticillioides nigrescens###Colony diameter (cm)###Inhibition (%)

Stenotrophomonas maltophilia###2.60###2.78###1.92###1.60###1.52###2.08###76.84 A

Pseudomonas ultimum###8.56###3.34###2.04###2.42###1.56###3.58###60.18 B

P. fluorescens###8.86###4.02###2.40###3.30###1.50###4.20###55.38 D

P. putida###8.18###7.40###3.54###3.36###1.56###4.81###46.58 F

P. aeruginosa###8.68###3.30###3.46###2.26###1.52###3.84###57.29 C

Bacillus subtilis###7.72###2.86###2.82###2.84###1.52###3.55###60.53 B

B. megaterium###8.38###3.32###2.96###2.96###1.78###3.88###56.89 C

B. cereus###8.74###7.08###1.94###1.92###4.76###4.89###45.69 F

B. thuringiensis###8.86###7.48###1.82###1.78###2.82###4.55###49.42 E

Control###9.00###9.00###9.00###9.00###9.00###9.00###0.00 G

This was followed by B. megaterium where disease index was 18.67%, while the other two bacterial biocontrol agents i.e., decreased disease index to 20.0-20.89%, respectively with no significant difference between them (Table 9; Fig. 4).

Effect on potted plants: Treatment with S. maltophilia, (curative), which was the most effective in the detached leaves assay, to dracaena (cv. Bicolor) previously inoculated with dracaena leaf spot fungi, significantly decreased mean percentage of infection with leaf spots to 29.33% compared to 100% for the inoculated untreated control. More pronounced effect was observed for disease index as S. maltophilia decreased disease index to 7.6% compared to 83.6% for the inoculated untreated control (Table 10).

Efficacy of Fungal Biocontrol Agents

In vitro effect on colony growth of dracaena leaf spot fungi: All the tested Trichoderma spp., i.e. Trichoderma harzianum, T. hamatum and T. album obviously inhibited growth diameter of the tested dracaena leaf spot fungi. Data in Table (11) showed that percentage of colony diameter inhibition ranged between 64.22% and 75.02%, where T. harzianum showed the highest inhibition effect. This was followed by T. hamatum and T. harzianum where percentages of inhibition were 67.73% and 64.22%, respectively (Table 11).

In vitro effect on spore germination and germ tube: All Trichoderma fungal bioagents significantly inhibited spore germination of the tested dracaena leaf spot fungi and percentage of inhibition ranged between 48.00% with T. album to 30.00% with T. harzianum (Table. 12). Meantime, the different biocontrol agents of Trichoderma spp. significantly decreased length of germ tube of the germinated spores of the different fungi (Table 12). The T. album was the most effective as exhibited mean inhibition effect on the tested leaf spot fungi as high as 90.21%. This was followed by T. hamatum and T. harzianum, where inhibition effects were 84.56% and 80.33%, respectively (Table 12).

In Vivo Effect of Trichoderma on Dracaena Leaf Spot Disease

Effect on detached leaves: Data presented in Table (13), illustrated in Fig. 5 showed that treatment with Trichoderma spp. (curative) significantly decreased percentages of infection and disease index of leaf spots developed on detached leaves of dracaena (cv. Bicolor), previously inoculated with the tested five dracaena leaf spot fungi . The T. album showed the highest effect and decreased percentage of infection to 35.54%, compared to 92.80% in the inoculated untreated control and also, decreased the developed disease index to 9.34% compared to 100% for the inoculated, untreated control.

Effect on potted plants: Data presented in Table (14), illustrated in Fig. (6) showed that treatment with T. album (curative), which exhibited the highest effect in the detached leaves assay, to potted plants of dracaena (cv. Bicolor) inoculated with five dracaena leaf spot fungi significantly decreased mean percentage of infection to 19.99% compared to 100% for the inoculated untreated control. Meanwhile, T. album decreased the developed disease index to 4.93% compared to 83.60% for the inoculated untreated control (Table 14; Fig. 6).

Table 8: The in vitro percentage of spore germination, length of germ tube and inhibition of five pathogenic dracaena leaf spot fungi treated with nine bacterial biocontrol agents

Leaf###spot###Spore Germination (%)###Germ tube length (um)

fungi Bacterial Botryodiplo Fusarium###Fusarium###Fusarium###Gibellulo Mean###Botryodipl###Fusarium###Fusarium###Fusarium###Gibellulo###Mean

Bio agents###dia###graminear###proliferat verticillioi psis###inhibition###odia###graminear###proliferat verticillioi psis###inhibition

###theobromae um###um###des###nigrescens (%)###theobromae###um###um###des###nigrescens (%)

Stenotrophomonas 40.00###50.00###60.00###58.00###48.00###48.80A###0.10###0.05###0.15###0.14###0.07###88.77A

maltophilia

Pseudomonas###66.00###62.00###66.00###62.00###64.00###36.00B###0.15###0.15###0.20###0.20###0.17###80.45C

ultimum

Pseudomonas###70.00###68.00###80.00###80.00###86.00###23.20D###0.23###0.25###0.28###0.39###0.37###65.65E

fluorescens

Pseudomonas###68.00###72.00###72.00###74.00###66.00###29.60C###0.23###0.27###0.22###0.22###0.30###72.15D

putida

Pseudomonas###74.00###64.00###80.00###76.00###60.00###28.00C###0.23###0.22###0.21###0.22###0.31###73.00D

aeruginosa

Bacillus###56.00###60.00###48.00###56.00###60.00###44.00A###0.10###0.13###0.10###0.14###0.21###84.97B

subtilis

Bacillus###76.00###74.00###66.00###72.00###68.00###28.80C###0.21###0.22###0.17###0.26###0.30###73.88D

megaterium

Bacillus###76.00###68.00###72.00###76.00###72.00###27.20C###0.23###0.33###0.30###0.24###0.35###67.15E

cereus

Bacillus###72.00###72.00###80.00###70.00###86.00###24.00D###0.27###0.26###0.31###0.24###0.35###67.15E

thuringiensis

Control###100.00###100.00###100.00###100.00###100.00###0.00E###0.77###1.07###0.80###0.95###0.85###00.00F

Table 9: The in vivo effect of four bacterial biocontrol agents (curative) on disease incidence and disease index on detached leaves of dracaena (cv. Bicolor) inoculated with five dracaena leaf spot fungi

###Disease incidence###Disease index (%)

Dracaena leaf###Infected and treated###Infected###Infected and treated###Infected

spot fungi###S. maltophilia###P. ultimum###B. subtilis B. megaterium###untreated###S. maltophilia###P. ultimum B. subtilis###B. megaterium###untreated

Botryodiplodia 66.67###88.89###88.89###66.67###100###26.67###28.89###31.11###17.78###90.00

theobromae

Fusarium###66.67###77.78###88.89###88.89###100###15.56###31.11###24.44###24.44###96.00

graminearum

Fusarium###33.33###55.56###55.56###44.44###100###11.11###11.11###13.33###13.33###92.00

proliferatum

Fusarium###44.44###77.78###66.67###66.67###100###13.33###22.22###15.56###22.22###94.00

verticillioides

Gibellulopsis###55.56###66.67###77.78###55.56###100###13.33###11.11###15.56###15.56###92.00

nigrescens

Mean###53.33D###73.33B###75.55B###64.44C###100 A###16.00D###20.89 B###20.00 B###18.67C###92.80 A

Table 10: Disease incidence and index of leaf spots developed on dracaena plants (cv. Bicolor) inoculated with five pathogenic leaf spot fungi and sprayed (curative) with S. maltophilia bacterial biocontrol agent

Dracaena leaf spots fungi###Percentage of infection (%)###Disease index (%)

###Infected and treated###Infected untreated###Inhibition (%)###Infected and treated###Infected untreated###Inhibition (%)

Botryodiplodia theobromae###36.67###100###63.33 D###10.7###82.00###86.95 B

Fusarium graminearum###33.3###100###66.67 CD###10.0###90.00###88.88 AB

Fusarium proliferatum###20.0###100###80.00 A###4.7###80.00###94.12 A

Fusarium verticillioides###26.7###100###73.33 A###6.7###84.00###92.02 AB

Gibellulopsis nigrescens###30.0###100###70.00 BC###6.0###82.00###92.68 AB

Mean###29.33###100###70.67###7.60###83.60###90.93

Discussion

Dracaena species are well known in Egypt and other parts of the world as an important indoor ornamental plants as well as important medicinal plants for curing a number of diseases (Wagih et al., 1989; Jia-Yi et al., 2014).

Unfortunately, dracaena plants are attacked by several plant diseases particularly the fungal leaf spots, which constitute a threat to dracaena cultivation and industry in Egypt and several parts of the world (Abd El-Zaher et al., 2005; Thongkantha et al., 2008).

For controlling dracaena leaf spot fungi, six common fungicides, namely Dithane M45(r) 80% WP, Kocide 2000(r) 53.8% WP, Ridomil Gold Plus (r)42.5% WP, Score(r) 25 % E.C, Equation Pro(r) 52.5% WG and Kemazed(r) 50% WP were investigated in vitro and in vivo. All the tested fungicides significantly decreased the in vitro growth of the tested dracaena leaf spot fungi. Percentage of inhibition on colony diameter by the tested fungicides ranged between 49.64% with Dithane M45(r) to 82.18% with Score(r) which was followed by Kemazed(r) (69.15%). Also, inhibition of spore germination was the highest (72.8%) by Dithane M45(r) while inhibition of germ tube reached 89.91% with Kemazed(r).

The in vivo tests using the detached leaves assay as well as the inoculation of potted plants of dracaena (cv. Bicolor) supported the in vitro results. Kemazed(r) decreased disease incidence of leaf spots in potted plants to 16.67% with 4.1% disease index compared to 100% and 83.6%, respectively for the untreated inoculated control. These results were in harmony with those obtained by Chase (2010), Siddique et al. (2014). The high positive efficacy of fungicides against fungal growth and their means of reproduction as well as the disease incidence and severity may ascribed to their toxic nature to fungal structure and its physiological activities, and to their long period of persistence on plant surfaces (Gill and Garg, 2014).

The bacterial and fungal biocontrol agents proved to be an effective means for controlling the dracaena leaf spot fungi (Heydari and Pessarakli, 2010). The in vitro assays conducted showed that the bacterial biocontrol agents i.e., S. maltophilia, P. ultimum, P. fluorescens, P. putida, P. aeruginosa, B. subtilis, B. megaterium, B. cereus and B. thuringiensis, significantly decreased colony growth diameter (76.84-45.69%) of dracaena leaf spot fungi as well as spore germination (48.80-23.20%) and length of the germ tube (88.77-65.65%) of the germinated spores compared to the untreated inoculated control. Treatment with S. maltophilia showed the most consistent highest inhibition effect in this respect, followed by treatments with P. ultimum, B. subtilis, and B. megaterium, respectively.

Meantime, the results of the in vivo tests on dracaena (cv. Bicolor) using the detached leaves method and the potted plants technique supported the in vitro results. S. maltophilia showed the highest effect as decreased disease incidence and disease index to 29.33% and 7.6%, respectively compared to 100% and 83.6% for the untreated inoculated control in the potted dracaena plants (cv. Bicolor) for the above mentioned disease parameters, respectively. These results were in agreement with those of Asaka and Shoda (1996); Liu and Wenshi (1997); Oraghi et al. (2011); Palaniyandi et al. (2011) and Abdalla et al. (2014). This could be explained in view of Kloepper and Schroth (1981) and Sivasakthi et al. (2014) that bacterial bioagents promoted plant growth, increased availability of mineral nutrients and produced plant-growth regulators that may be responsible for better healthy plants and consequently less disease parameters.

Table 11: The in vitro colony diameter and inhibition of five dracaena leaf spot fungi on PDA amended with three fungal biocontrol agents of Trichoderma spp

Leaf spot fungi Fungal###Botryodiplodia###Fusarium###Fusarium###Fusarium###Gibellulopsis###Overall mean

bio agents###theobromae###graminearum###proliferatum###verticillioides###nigrescens

###Colony diameter###Colony diameter###Colony diameter###Colony diameter###Colony diameter###Colony diameter (Cm) Inhibition (%)

Trichoderma harzianum###3.18###1.70###1.52###2.84###2.00###2.25###75.02 A

T. hamatum###4.28###2.26###1.60###3.44###2.94###2.90###67.73 B

T. album###5.26###1.44###1.84###3.76###3.80###3.22###64.22 C

Control###9.00###9.00###9.00###9.00###9.00###9.oo###0.00 D

Table 12: Percentage of spore germination, length of germ tube and inhibition of five dracaena leaf spot fungi treated with three fungal bio agents

Fungi###Spore Germination (%)###Germ tube length (um)

Fungal bio Botryodiplodia Fusarium###Fusarium###Fusarium###Gibellulopsis Mean###Botryodiplodia Fusarium###Fusarium###Fusarium###Gibellulopsis###Mean

agents###theobromae###graminearum proliferatum verticillioides nigrescens###inhibition theobromae###graminearum proliferatum verticillioides nigrescens###inhibition

###(%)###(%)

Trichoderma 60.00###70.00###80.00###70.00###70.00###30.00C###0.12###0.12###0.16###0.18###0.22###80.33C

harzianum

T. hamatum###60.00###60.00###70.00###60.00###70.00###36.00B###0.13###0.11###0.15###0.11###0.18###84.56B

T. album###60.00###50.00###40.00###50.00###60.00###48.00A###0.10###0.11###0.07###0.06###0.10###90.21A

Control###100.00###100.00###100.00###100.00###100.00###0.00D###0.79###1.05###0.77###0.95###0.89###0.00D

Table 13: Disease incidence and disease index of leaf spots developed on detached leaves of dracaena (cv. Bicolor) inoculated with five pathogenic dracaena leaf spot fungi and treated (curative) with two Trichoderma spp

Dracaena leaf spot fungi###Percentage of infection (%)###Disease index (%)

###Inoculated and treated###Inoculated and treated

###T. harzianum###T. album (Bio Zeid)###Inoculated untreated###T. harzianum T. album (Bio Zeid)###Inoculated untreated

Botryodiplodia theobromae###66.67###55.56###100###22.22###15.56###90.00

Fusarium graminearum###55.56###44.44###100###17.78###11.11###96.00

Fusarium proliferatum###44.44###22.22###100###8.89###6.67###92.00

Fusarium verticillioides###55.56###33.33###100###11.11###8.89###94.00

Gibellulopsis nigrescens###33.33###22.22###100###6.67###4.44###92.00

Mean###51.12 B###35.54 C###100.00 A###13.34 B###9.34 C###92.80 A

Table 14: Percentage of infection and disease index of leaf spots developed on potted plants of dracaena (cv. Bicolor) inoculated with five pathogenic dracaena leaf spot fungi and treated (curative) with Trichoderma album(Bio Zeid)

Dracaena leaf spot fungi###Disease incidence###Disease index (%)

###Inoculated and treated###Inoculated and treated

###Trichoderma album. (Bio Zeid)###Inoculated untreated###Trichoderma album. (Bio Zeid)###Inoculated untreated

Botryodiplodia theobromae###26.70###100###8.70###82.00

Fusarium graminearum###23.30###100###5.30###90.00

Fusarium proliferatum###13.30###100###2.70###80.00

Fusarium verticillioides###16.70###100###4.00###84.00

Gibellulopsis nigrescens###20.00###100###4.00###82.00

Mean###19.99 B###100.00 A###4.93 B###83.60 A

Also, Bacillus and Pseudomonas genera are among the plant growth promoting rhizobacteria PGPR. The PGPR could act as biocontrol agents by production of antibiotics, triggering induced local or systemic resistance, or preventing the deleterious effects by acting as rhizoremediators (Glick et al., 2007). Meanwhile, Members of Bacillus spp. were able to produce various lytic enzymes e.g., chitinase and b-1, 3-glucanase (Pankaj and Dubey, 2012), along with induction of systemic resistance of plants, such increasing the activities of plant defense related enzymes of peroxidase, polyphenol oxidase and phenylalanine ammonialyase (Jayaraj et al., 2004). Also, P. fluorescens was able to produce and secrete secondary metabolites that might play a role against activity and spore germination of plant pathogens (Sivasakthi et al., 2014).

On the other hand, The in vitro tests of three Trichoderma spp. i.e., T. harzianum, T. hamatum and T. album significantly inhibited colony growth diameter and spore germination of the tested dracaena leaf spot fungi. T. harzianum, however, showed the highest inhibition on colony diameter (75.02%) while, T. album was the most effective to inhibit spore germination (48%) and germ tube (90.21%) of the germinated spores. The in vivo assays of the fungal biocontrol agents supported the in vitro obtained results. Using the inoculation of the potted plants technique, T. album (Bio-Zeid) showed the highest effect as decreased percentage of infection and disease index to 19.99% and 4.93% compared to 100% and 83.6%, respectively for the untreated inoculated control. This could be explained in view that, the Trichoderma spp. could protect the plant by establishing themselves in the rhizosphere (root zone) and establish a barrier against pathogen attack (Hermosa et al., 2012).

Also, early applications of Trichoderma spp. protected plant roots by removing secreted nutrients that pathogens might use (Azarmi et al., 2011). Meantime, many plant pathogens contain chitin as a component of their cell wall. Trichoderma release chitinases that have been shown to disrupt the cell wall of these pathogens. These enzymes dissolve the cell wall and create holes in the pathogen. Once damaged, the pathogen itself becomes the prey of other soil microflora (Hermosa et al., 2012). Also, it has been shown that hyphae of T. aureoviride isolate grew and coiled around the hyphae of F. oxysporum as well as an intense vacuolation could be observed within the cytoplasm of the pathogens hyphae involved (Brzezinska, et al., 2014). Also, it has been suggested that Trichoderma produced extracellular b-(1-4) glucanase and chitinase, which are key enzymes in the lysis of fungal cell walls (Sharma and Trivedi, 2010).

The active biocontrol agents such as species of Trichoderma have evolved numerous mechanisms that are involved in biological control. The mechanisms include competition for space and nutrients, antibiosis, antagonism, inhibition of pathogen enzymes and plant growth stimulation (Harman et al., 2004; Abdel-Khair et al., 2010). Also, different foliar diseases of plant were effectively controlled with each or more species of these bioagents when they were sprayed on susceptible plants (Bartmanska and Gladysz, 2006; Hilal et al., 2009; Ragab et al., 2011).

Conclusion

The obtained results surely can help to establish a sustainable, eco-friendly strategy for controlling the fungal leaf spots of dracaena.

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