Journal of Phytopathology and Pest Management 8(1): 92-105, 2021

pISSN: 2356-8577 eISSN: 2356-6507

Journal homepage: http://ppmj.net/

∗

Corresponding author:

Hoda A. M. Ahmed,

E-mail: hudafatah@yahoo.com

92

Efficacy of biological therapies against onion basal rot

caused by Fusarium oxysporum f. sp. cepae under field

and storage conditions

Hoda A. M. Ahmed1*, Zeinab Soliman2, Mohamed A. Khalil1, Sayed B. M. Fawaz1

1Plant Pathology Research Institute, Agricultural Research Center, Giza, Egypt

2Assuit University Moubasher Center (AUMMC), University Assiut, Egypt

Abstract

Keywords: Allium cepa, basal rot, antagonism, Fusarium oxysporum f. sp. cepae, yeasts.

Basal rot disease of onion is caused by Fusarium oxysporum f. sp. cepae (Hans.)

(FOC) economically significant losses wherever onion is grown. Fusarium

oxysporum were isolated from diseased onion cultivated in different places of

Assiut, Egypt. Efficacy of certain yeasts was evaluated for controlling the basal

rot of onion in vitro. Among of the tested isolates, Saccharomyces cerevisiae gave

the greatest inhibition (57.74%) and Candida tropicalis (1) significantly exerted

the greatest reduction of mycelial growth of F. oxysporum f. sp. cepae (51.18%).

Based on the in vitro screening, effective yeasts were evaluated under greenhouse,

field and storage conditions. Yeasts were applied by two methods (add the

pathogen and antagonistic yeasts to soil before sowing seedling onion, and seedling

onion soaking in yeasts for 20 minute). Both methods of inoculation showed

substantial impact on disease development and plant growth. Add method caused

maximum reduction in plant germination, followed by soaking method as

compared to control. Application of fungicide (Captain) as compared brought a

remarkable increase in seedling emergence of treated plants inoculated with F.

oxysporum as compared to the untreated plants. In conclusion, tested yeasts were

useful as an alternative to chemical control of the onion basal rot and to enhanced

growth and yield of onion.

Ahmed Hoda et al., 2021

93

1. Introduction

Onion (Allium cepa L.), is one of the oldest

vegetables, has been used as spice and

medicine for thousands of years (Keusgen,

2002). Fusarium basal rot of onion is an

economically important disease to which onion

bulbs and shallots are sensitive during all their

growth stages (Cramer, 2000). To manage the

disease, chemical control is very effective, but

it is not economical and pollutes the

environment. Use of resistant cultivars is

another acceptable strategy of control however

onion cultivars with acceptable level of

resistance are limited. Researchers have

recently considered biological control as a

complementary approach for controlling this

disease. Yeasts treatments were suggested to

play a beneficial role in cell division and cell

enlargement (Freimoser et al., 2019). yeasts

were demonstrated to be effective biocontrol

agents of seedlings onion post-emergence

damping off. Various yeast species have been

reported as active biological control agents.

Some effectively saccharomyces cerevisiae

was used as bioagents root rot pathogens under

greenhouse conditions (Abd El-kader et al.,

2012). Candida glabrata and C. maltosa

significantly reduced the incidence of late

maize wilt disease when applied by seed

inoculation (El-Mehalawy et al., 2004). Pichia

guilliermondii gave hence possess potential to

control wilt disease in tomato crop (Nguyen et

al., 2011). Shalaby and El-Nady (2008) found

that seed soaking, or soil inoculation with S.

cerevisiae increased germination rate, survival

of plants and reduction of pre and post

emergence damping off and inhibited

Fusarium oxysporum liner growth in vitro.

Also, they found that pre- and post- emergence

damping off was reduced significantly when

seeds of faba bean were coated with a water

suspension of the yeast (109 CFU ml-1). The

beneficial effects of these microorganisms last

longer than that of chemicals and can therefore

protect the plant throughout all growth stages.

Under greenhouse and field conditions, control

of plant diseases using antagonistic yeasts can

be effective. Also, biological control can limit

the instances of basal rot of onion caused by F.

oxysporum and reduced probability of disease

development. If pathogen attacks the host plant

late in the season, the symptoms may not

appear until onion bulbs are in storage (Ozer et

al., 2003) Present study found that biological

agents inhibit the growth of F. oxysporum.

Fusarium oxysporum f. sp. cepae (Hanz) Snyd

attack onion bulbs in storage and cause rotting

of onion bulbs during storage. In storage, onion

bulbs appear spongy or sunken, infected bulbs

are softened, brown and watery when cut open

during storage bulbs are affected by many

microorganisms leading to rot being

commercially important crop of the state; it

was felt necessary to carry out investigations

on storage rot of onion. Despite the

achievement in production technology and

availability of good varieties of onion, the post

harvest losses during storage is still an ailing

cause which leads to significant qualitative and

quantitative losses during storage up to 25-

30%. The onion postharvest losses were

estimated worth Rs 600 crores is found to be

due to desiccation, decay and sprouting,

(Kumar et al., 2015). The rationale behind such

post-harvest losses till today is the

unavailability of good storage facilities during

post-harvest storage phase. There seems a big

gap between the storage facility and the storage

capacity which is ultimately leading to the

unforeseeable postharvest decay and

deterioration of onion bulbs. Therefore, the

main objectives of this study were attempted to

apply a of some antagonistic yeasts, enhanced

the biocontrol of onion basal rot and studied

the suppressive effect of some free yeast

strains against Fusarium oxysporum, under

greenhouse, field and storage conditions.

2. Materials and methods

2.1 Isolation of the causal pathogen of onion

basal rot disease

Bulb samples of onion showing typical basal

Ahmed Hoda et al., 2021

94

rot symptoms were collected from different

locations in the Assiut governorate of Upper

Egypt. Isolation of the pathogen from the

infected bulbs was performed by the tissue

segment method described by Rangaswami

(1958). Infected portions of bulb samples were

cut into small pieces and washed thoroughly in

tap water. The pieces were then disinfected by

immersing in 1% sodium hypochlorite (SH)

solution for one minute, rinsed three times in

sterilized distilled water (SDW), and dried

between folds of sterilized filter papers.

Disinfected pieces were transferred aseptically

into Petri plates containing Potato Dextrose

Agar (PDA) medium supplemented with 400

mg streptomycin sulfate per liter of medium.

The plates were then incubated at 25±0.5 °C

for 5 days and examined daily for fungal

growth. The growing fungal colonies were

purified by single spore and hyphal tip

techniques, followed by sub-culturing onto a

freshly prepared PDA medium at the same

conditions. According to cultural and

microscopical characteristics of colony

mycelia and spores, isolated fungi were

morphologically identified (Leslie &

Summerell, 2006; Gerlach & Nirenberg,

1982). Pure cultures of isolated fungi were

maintained at 5 °C on PDA slants for further

studies.

2.2 Pathogenicity test

The pathogenic capability of all fungal isolates

obtained to cause basal rot disease was

investigated on the onion Giza 6 cultivar under

greenhouse conditions. The inoculum of each

isolate tested was prepared by placing two

disks (0.7 cm in diameter) taken from the 7-

day-old culture onto an autoclaved sand-maize

medium (80 g sieved fine river sand, 20 g

maize meal, and 50 ml water) in conical flasks

tightly closed with cotton plugs. Then flasks

were incubated at 27±0.5 °C for 14 days.

Formalin-sterilized plastic pots (35 cm in

diameter) were filled with autoclaved loam

soil (5.0 kg of each), infested with 150 g

inoculum of each isolate tested, and then

slightly irrigated every other day for a week.

Pots treated with the same amount of non-

inoculated sand-maize medium served as

control. Another week later, onion seedlings

were disinfected by dipping in 1% SH solution

for 3 min, rinsed three times in SDW for 10

min, and then transplanted at a rate of 6

seedlings per pot. The experiment was

performed with four pots (replicates) of each

isolate tested in a completely randomized

design. Then pots were checked daily and

irrigated when necessary. Observations of

basal rot symptoms were recorded daily and

continued with disease development until the

whole plant's complete rotting. Six months

after planting, plants were uprooted, and

percentages of disease incidence (DI) and

surviving plants (SP) were calculated

according to the following formula:

𝐷𝐼 % = 𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑑𝑖𝑠𝑒𝑎𝑠𝑒𝑑 𝑝𝑙𝑎𝑛𝑡𝑠

𝑇𝑜𝑡𝑎𝑙 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑝𝑙𝑎𝑛𝑡𝑠 × 100

𝑆𝑃 % = 𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑠𝑢𝑟𝑣𝑖𝑣𝑖𝑛𝑔 𝑝𝑙𝑎𝑛𝑡𝑠

𝑇𝑜𝑡𝑎𝑙 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑝𝑙𝑎𝑛𝑡𝑠 × 100

2.3 Preparation and growth conditions of

used yeast strains

Pure cultures of yeast strains were previously

isolated from different locations in Egypt and

identified according to classical methods used

in yeast taxonomy (van der Walt & Yarrow,

1984). Yeast strains which exhibit the highest

antagonistic effects against the pathogenic F.

oxysporum were selected and cultured on

glucose peptone yeast extract (GPY) medium

(Difco, 1985) by shaking on a rotary shaker at

28±0.5 °C for two days.

Ahmed Hoda et al., 2021

95

2.4 Study of antifungal activity of yeast

strains against FOC

Nineteen yeast strains were tested for the

antifungal activity against FOC using the

direct dual culture technique. Yeast strains

were initially grown on GPY broth in test tubes

by shaking on a rotary shaker at 28±0.5 °C for

2 days, as mentioned before. On the GPY

plates' surface, 0.1 ml of each aqueous yeast

suspension with a concentration of 106 cells

ml-1 were plated. Then yeasts were grown for

5-7 days as a lawn on the Sabouraud agar

surface, whereby 5 mm discs in diameter were

cut. Disks from each yeast strain were placed

on a PDA medium on one side of the Petri

plate, and the opposite side at an equal distance

was inoculated by 5 mm- disks of FOC isolate

taken from 7-days-old culture. Four plates

were used for each yeast strain tested, and the

inoculated plates with FOC discs only were

served as control. The entire experiment was

twice repeated for confirmation of the results.

After 10 days of plate incubation at 28±0.5 °C,

antifungal activity was determined by

measuring the zone of inhibition (cm)

produced by yeasts against FOC. Also, the

located mycelium inside the zones of

inhibition was microscopically examined.

Growth inhibition (%) of FOC was calculated

using the following formulae:

𝐺𝐼 % = 𝐶 − 𝑇

𝐶 × 100

Where GI = growth inhibition, C = mycelium

radial growth in control, and T = mycelium

radial growth produced by yeast strain tested.

2.5 Evaluation of antagonists under

greenhouse conditions

Yeast strains that showed the highest

suppression rate against FOC in vitro were

applied to control the disease under

greenhouse and field conditions. Pots of 35 cm

diameter were filled with sterilized soil mixed

with the FOC inoculum one week before

planting. Soil infestation was conducted 7 days

before sowing by adding 3% of the inocula

with the soil in each pot. All treatments were

applied by two methods the first was add the

pathogen and antagonistic fungus (yeasts) at

the rates of 106 CFU/g were mixed with soil

pots before sowing seedling onion, the second

methods seedling onion soaking in yeasts for

20 minutes. Seedling onion treated in

fungicide Captain (50% WP) was used as a

reference. Each pot was planted with 5

seedling onion Giza 6 cultivar. Four replicates

were used to each treatment and pots

containing inoculum of FOC only were used

as control. Plants were irrigated when

necessary and examined periodically. Data

disease incidence and plant mortality were

recorded after 6 months of seed sowing. The

formulae used to determine the diseases

incidence were followed as mentioned before.

2.6 Evaluation of antagonists under field

conditions

A field experiment was conducted to evaluate

the efficacy of biocontrol agents on onion

basal rot during 2019/2020 and 2020/2021

seasons. Seedlings of onion cv. Giza 6 were

planted in rows in plots size 5 × 4 m and

spacing 15 cm. A Randomized Block Design

(RBD) was used with three replications.

Before sowing, the treatments were applied by

two methods such as greenhouse treated, and

fungicide Captain was used for comparison.

All normal agronomical practices including

irrigation, weeding and fertilizer application

were followed at regular intervals. Natural

incidence of basal rot of onion was recorded.

At harvest time, plant samples (10 healthy

plants each) were taken at random from each

plot to determine the growth parameters, plant

Ahmed Hoda et al., 2021

96

height (cm), bulb diameter (cm), Average bulb

weight (g) and total bulb yield (ton/feddan)

were recorded (feddan = 4200 m² = 0.420

hectares = 1.037 acres).

2.7 Bulb storage

After harvest, 5 kg onion bulbs for each

replicate from treatments were placed in nylon

mesh onion bags without removing the stalks

and Store in a well-ventilated place. During

storage the air was continuously drawn over

the onions by means of a fan to remove excess

moisture. The bulbs were stored at low

temperature for 30, 60, or 90 days. After the

appropriate duration of storage, five bulbs

were removed from each replicate bag and

evaluated for severity of Enterobacter bulb

decay. Each onion bulb was sliced from the

neck to the basal plate, and the cut surface area

of the fleshy scales was rated visually for

severity of Enterobacter bulb decay

(percentage of the surface area of the fleshy

scales showing symptoms typical of this

disease) (Schroederand du Toit, 2010). A low

incidence of bulbs (1 to 5%) showed

symptoms characteristic of other storage rots

(e.g., neck rot caused by Botrytis aclada and

B. allii, black mold caused by Aspergillus

niger, Fusarium basal rot caused by Fusarium

oxysporum f. sp. cepae.

2.7 Statistical analysis

Analysis of variance (ANOVA) was carried

out using MSTAT-C program. The least

significant difference (LSD) at P≤0.05 was

applied to detect differences among treatments

(Gomez and Gomez, 1984).

3. Results and Discussion

3.1 Pathogenicity test

Nine isolates of Fusarium oxysporum f. sp.

cepae were isolated from different localities of

Assiut governorate in Egypt. Identification of

the isolates was based on spore morphology

and culture characteristics, as described by

Booth (1985) and Schwartz (2010) (Figure 1).

Figure 1: Fusarium oxysporum f. sp. cepae; A: colony on DPA after 7 days, B,

C and D: hyphae bearing short monophialides, E: micro- and macroconidia.

Pathogenicity test of isolates are presented in

Table (1) indicate that all isolates proved to be able to infect onion plants causing basal rot

symptoms. Typical symptoms observed were

Ahmed Hoda et al., 2021

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yellowing and eventual dieback of the leaves,

drying of leaves and in advance stage, basal

portion of plants completely rotted and entire

plant got collapsed on ground, which was

noticed 15 days after transplanting in the pots.

Uninoculated plants remained healthy. These

results agree with Saxena (2007) isolated

Fusarium oxysporum f. sp. cepae from

infected bulb in USA. Davis (2008) reported

the pathogen, F. oxysporum f. sp. cepae

causing onion and garlic basal rot.

Dissanayake et al. (2009) studied the

pathogenicity of 32 isolates of Fusarium spp.

on five commercial cultivars of welsh onion

and proved that five Fusarium oxysporum

isolates had higher virulence to Allium spp.

Table 1: Pathogenic capability of F. oxysporum f. sp. cepae on onion cultivar Giza 6.

Isolates number

Disease incidence (%)

Surviving plants (%)

1

41.66

58.33

2

45.83

54.16

3

50.00

4

66.66

33.34

5

29.16

70.83

6

33.33

66.67

7

37.49

62.50

8

20.82

79.17

9

79.16

22.22

Control

0.00

100.00

L.S.D. at 5%

11.41

2.77

3.2 Evaluation of yeast isolates as biocontrol

agents against the causal pathogen in vitro

Yeast isolates from different sources were

screening for their abilities to inhibit the

growth of the plant pathogen F. oxysporum.

These isolates were Candida tropicalis,

Cryptococcus albidus, Debaryomyces

hansenii, D. pseudopolymorphus,

Galactomyces candidus, G. pseudocandidus,

Klyuveromyces marixianus, Lachancea

thermotolerans, Papiliotrema laurentii, Pichia

caribaea, Saccharomyces cerevisiae, and

Saccharomyces sp., which were identified by

using morphological and physiological

characters according to Barnett et al. (2000)

and molecular by the internal transcribed

spacer (ITS) sequences of nuclear ribosomal

DNA. The studied yeast nineteen isolates were

shown variable antagonistic impact against the

plant pathogen F. oxysporum f. sp. cepae

giving inhibition percentages of the pathogen

growth ranging from 22.28 to 57.74 (Table 2

and Figure 2). The strains Saccharomyces

cerevisiae, Candida tropicalis, Pichia

caribaea and Saccharomyces sp. were

exhibited the highest antagonistic effect.

These promising strains were selected for the

applied experimental studies in greenhouse

and field.