Investigation and comparison of the ice nucleation activity in Fusarium avenaceum and Fusarium...

International Journal of Farming and Allied Sciences Available online at www.ijfas.com

©2014 IJFAS Journal-2014-3-5/518-528/ 31 May, 2014

ISSN 2322-4134 ©2014 IJFAS

Investigation and comparison of the ice nucleation activity in Fusarium avenaceum and Fusarium

acuminatum

Tahereh Seifi1, Saghar Ketabchi1, Heshmatollah Aminian1, Hassan Reza Etebarian1 and Mohammadreza Kamali2*

1. University of Tehran, College of Aboureyhan, Department of Plant Protection, P.O.Box: 11365/7117 Tehran, Iran

2. University of Aveiro, Department of Environment and Planning, P.O. Box 3810-193 Aveiro, Portugal

Corresponding author: Mohammadreza Kamali

ABSTRACT: n spite of providing some industrial advantages, formation of the natural and artificial ice nuclei is considered as one of the most important causes limiting the plant growth and hence, incurring the economic and ecological issues, especially in tropical and subtropical regions. Hence, studying this feature in heterogeneous and homogeneous particles can considerably help to control the adverse effects of the ice nucleation on the crop production via, for instance, producing the negative mutants to prevent frostbite and hypothermia of plants. The present manuscript aims to investigate the ice nucleation activity in some fungal microorganisms. For this, several Fusarium species were isolated from the suspected plants and identified and then a set of tube nucleation tests was conducted in order to investigate the potential of the ice nucleation activity in the identified species. Based on the obtained results, the drop freezing test was conducted on Fusarium avenaceum and Fusarium acuminatum species, which showed significant ice nucleation activity, using tube nucleation tests. In this regard, variables such as fungal suspension concentration, time duration, temperature and the effect of the heating at higher temperatures prior to cold treatment were considered in order to study the rate and severity of the ice nucleation activity in the above-mentioned fungal species. The obtained results indicated that F. avenaceum presents stronger ice nucleation activity compared with F. acuminatum. In addition, it seems that as the temperature decreases, differences between ice nucleation activities in these microorganisms are reduced. Eventually, at the temperatures below 7ـ

°C, both studied

microorganisms showed the severe ice nucleation activity. Keywords: Ice nucleation activity, Fusarium avenaceum, Fusarium acuminatum, Tube nucleation test, Drop freezing test

INTRODUCTION

When water cools under atmospheric pressure, it may remain liquid even at temperatures below 0 ˚C (Kobashigawa, 2005). This process is known as supercooling. However, a large number of particles with biological (heterogeneous) and non-biological (homogeneous) origin, including dust, plant substances and organic or inorganic compounds have been identified with the capability of the ice nuclei formation and hence, inducing the freezing in higher temperatures. Ice nucleation activity (INA) has led such particles to be used in various applications including food industry (e.g., Zhang, 2010), commercial cold dryers (e.g., Geidobler and Winter, 2013) and biomedical applications (e.g., Kandra and Devireddy, 2008). Moreover, aforementioned particles can penetrate into the air layers and play an important role in the natural and artificial processes such as cloud seeding, through

Intl J Farm & Alli Sci. Vol., 3 (5): 518-528, 2014

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the aerosolization process (Murray and Jensen, 2010), inducing the beginning of water freezing at higher temperatures. In INA microorganisms, ice nucleation activity is attributed to the existence of insoluble protein compounds in their outer membrane (Warren and Wolber, 1991), which provide certain conditions to convert the water molecules to the ice embryos (Attard, 2012). Production of such complicated compounds is considered as a genetic trait which can be observed in both biotic and abiotic INA microorganisms (Joly, 2013). Diatoms (Knopf, 2011), some bacterial species including the strains of Pseudomonas spp., Erwinia spp., Pantoea spp. and Xanthomonas spp. (Nejad, 2004; Travert, 1997) and some fungi such as Fusarium moniliforme var. subglutinans (Kieft, 1988; Pouleur, 1992; Tsumuki, 1995), due to having this feature, are able to begin the process of water freezing even at a temperature of 2ـ ˚C. However, this exclusive ability of INA microorganisms has led them to adversely affect the biological processes. For example, the role of some microorganisms in inducing the cold stress in plants can be mentioned in this regard. The most frost sensitive plants such as oneـyear herbaceous plants, flowers of perennial fruit trees, branch, fruit and trunk of many species of trees and forest trees such as eucalyptus do not have any special mechanism to tolerate freezing (Wisniewski, 1997). Such cold stresses related injuries (or death) of plants normally take place as a result of rapid cooling at temperatures between 2ـ ˚C and 10ـ ˚C, in addition to the gradual freezing between -15

◦C and -40

◦C (Ashworth, 1985). INA microorganisms, in addition to the early formation of ice

nuclei, can bear consequences such as loss of turgor pressure, vacuolization, interaction of cytoplasmic membrane equilibrium, vesiculation, reduction of cytoplasmic streaming and general impairment in organs (Edwards, 1994). As a result, they may lead to economic and ecological problems through causing damage or destruction to plants (Lindow, 1982). As the first study on the fungal ice nucleation activity in Iran, the present manuscript aims to study this feature in some Fusarium species. As such activities were not observed in all studied strains, F. avenaceum and F. acuminatum, which are pathogens with global spread, were selected for further investigations. Then, the effects of various fungal suspension concentrations, time durations and temperatures as well as the heating at higher temperatures prior to cold treatment on the intensity of F. avenaceum and F. acuminatum ice nucleation activities were studied. Moreover, the differences between their performances at the same conditions have been surveyed.

MATERIALS AND METHODS

Microorganisms strains So far, active ice nuclei with biological origins have been isolated from plants, aerosols, precipitation and fresh water (Attard, 2012). In this study, plants suspected of infection with such microorganisms were used for isolation of Fusarium species. Licorice was taken as the main host of F. avenaceum and F. acuminatum was isolated from the plants of the Poaceae family (like wheat). Then, the samples were superficially sterilized. In this stage, the crown of the plants was divided into small pieces; these pieces were washed for 20 min under continuous flow of water and then immersed in 1.5 % sodium hypochlorite for 3 min; after 3 times of washing with distilled water and drying on a sterile paper filter, samples were transferred into petri dishes containing potato .agar medium (PDA)ـSome samples of plants are coarse or spongy and absorb disinfectant, destroying the pathogen. To resolve this problem, the samples were soaked in 95 % alcohol. The method of rubbing the cotton soaked in 95 % ethanol to contaminated samples was used to superficially disinfect the infected wooden parts in plants. Then, PDA medium was used for growth of the microorganisms and waterـagar (WA) medium and the method of the end of the row transmission were used for purification. To keep fungus, a certain amount of PDA medium was poured into test tubes and, after autoclaving, the contents of the tubes became solid in a diagonal form. Then, fungi were transferred into the tubes and incubated at a temperature of 25 ± 1 ˚C. After the fungi grew, the tubes were kept at a temperature of 4 ˚C (Fradkin and Patrick, 1985). In order to identify the genus of fungi, Barnett and Hunter key (Barnett, 1972) was used. Moreover, to identify the Fusarium species, PDA was used for culturing the studied strains. As a result, the detection of the colony shape and color and growth rate of the microorganisms was facilitated. Besides, to investigate the macroconidia and microconidia, carnation leafـagar medium (CLA) and synthetic nutrientـagar medium (SNA) were applied respectively as stated by Leslie and Summerell (2006). Microorganisms ice nucleation activity After culturing the microorganisms in a period of 28 days at 23

°C and a dark environment, first of all, a set of

tube nucleation tests (Pouleur et al., 1992) was conducted in order to find the presence or absence of ice

nucleation activity in the identified microorganisms. For this, about 2 cm2 of mycelium on the surface of the medium

was scratched and added to 1 mL of sterile 10 mM phosphate buffer (pH = 7) and then homogenized with a


520

homogenizer. Then, 40 µL of the suspension was prepared and added to 2 mL of sterile phosphate buffer, which

was cooled to -5 °C and kept at this temperature for 10 min. If the content of the tube was frozen, the related

microorganism was recorded as positive. For each isolate, tube nucleation test was carried out with four replicates.

The same stages were done with distilled water (two times of rectification instead of sterile phosphate buffer). In

cases where at least one tube showed a positive response, the experiments were conducted with more eight or

sixteen replicates in order to ensure. Thus, the isolates having ice nucleation activity were identified. Then, the drop

freezing test (Vali, 1971) was conducted with the aim of more precise investigation of the ice nucleation activity in

fungi which were recorded as positive INA, according to the results obtained from the tube nucleation tests. The

fungi, cultured on a PDA medium for 5 days and grown in darkness and a temperature of 23 °C, were used for this

test. Mycelium on the surface of the medium was scratched and added to 1 mL of sterile 10 mM phosphate buffer

(pH = 7) to reach the biomass concentration of 10 mg/mL (Measurement was done using a scale in a sterile

environment). The obtained suspension was totally homogenized by a homogenizer with glass blades and diluted

to a concentration of 1 mg/ml as described by Pouleur, (1992). In addition to concentrations of 1 mg/mL and 10

mg/mL, concentrations of 2 mg/mL and 5 mg/mL of fungal biomass were also used in the experiments. For this

purpose, sterile phosphate buffer (without fungus) was used as the control. 10 drops of 10 µL of each suspension

were removed and put on aluminum foils formed like a boat. The surface of the aluminum foil was already covered

by 1 % solution of paraffin in xylene and in order to remove the xylene, foils were kept at a temperature of 60 °C for

30 min. Then, aluminum foils were placed on the surface of waterـethanol (1:1 (v/v)) in a refrigerated bath unit.

Aluminum foils, on which drops of fungal suspension with a concentration of 1 mg/mL were poured, were kept in a

refrigerated bath unit at a temperature of 3ـ °C for 30 min. The number of frozen drops after 5 min, 10 min, 15 min,

20 min, 25 min and 30 min was recorded. The same measurements were performed at temperatures 4ـ °C, 5ـ

°C, 6ـ

°C, 7ـ

°C, 8ـ

°C and 9ـ

°C. In addition to the concentration of 1 mg/mL, concentrations of 2 mg/mL, 5 mg/mL and 10

mg/mL were also applied. It should be noted that the tests were done with sterile phosphate buffer as a control. All

above-mentioned drop freezing tests were repeated for three times. In order to evaluate the effects of the heating

at higher temperatures prior to cold treatment on fungal INA, the suspensions were faced with the temperatures of

23 °C (ambient temperature of the laboratory), 30

°C, 40

°C, 50

°C, 60

°C, 70

°C, 80

°C and 90

°C for 10 min and

then the suspensions were immediately cooled to 20 °C by putting it in a dish containing water and ice. Then, a set

of drop freezing tests was carried out as described before.

Statistical analyses

In order to investigate the ice nucleation activity in the studied microorganisms, the least significant difference

(LSD) method was applied to compare the means, using SPSS software. Moreover, factorial design was used for

analyzing the results obtained from the drop freezing tests. For this, the effect of the various suspension

concentrations was considered as factor (A) at five levels (0 mg/mL (control), 1 mg/mL, 2 mg/mL, 5 mg/mL and 10

mg/mL). In addition, six levels for the effect of the different time durations as factor (B) (5 min, 10 min, 15 min, 20

min, 25 min and 30 min), six levels for the effect of the various temperatures (4ـ °C, 5ـ

°C, 6ـ

°C 7ـ

°C, 8ـ

°C and 9ـ

°C)

as factor (C) and eight levels for the effect of the exposure of the microorganisms to higher temperatures (23 °C, 30

°C, 40

°C, 50

°C, 60

°C, 70

°C, 80

°C and 90

°C)

prior to cold treatment as factor (D) were considered.

RESULTS AND DISCUSSION

Microorganisms strains

After isolating the microorganisms from their main hosts, a total of 39 isolates were identified and tested for the

potential for ice nucleation activity. The list of the identified microorganisms has been provided in Table 1.


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Table 1. The obtained results from the drop freezing tests on the investigated microorganisms Water Test Results Buffer Test results

b Replicates

a Microorganisms

ـ( 4) ـ( 4) 8 Alternaria solani

ـ( 4) ـ( 4) 8 Alternaria solani

ـ( 4) ـ( 4) 8 Alternaria alternata

ـ( 4) ـ( 4) 8 Aspergillus spp.

ـ( 4) ـ( 4) 8 Verticillium dahliae






ـ( 4) (1)+ ,(3) ـ 8 Verticillium dahliae


ـ( 8) (2)+ ,(6) ـ 61 Penicillium spp.

ـ( 4) ـ( 4) 8 Penicillium spp.

ـ( 8) (1)+ ,(7) ـ 61 Trichoderma virens

ـ( 4) ـ( 4) 8 Macrophomina phaseolina

ـ( 4) ـ( 4) 8 Macrophomina phaseolina

ـ( 8) .Gaeumannomyces spp 61 (1)+ ,(7) ـ

ـ( 4) ـ( 4) 8 Gaeumannomyces spp.

ـ( 4) ـ( 4) 8 Magnaporthe salvinii

ـ( 4) ـ( 4) 8 Nattrassia spp.

ـ( 4) ـ( 4) 8 Rhizoctonia spp.

Fusarium oxysporum 61 (3)+ ,(5) ـ (1)+ ,(7) ـ

ـ( 4) ـ( 4) 8 Fusarium oxysporum



Fusarium oxysporum 44 (4)+ ,(8) ـ (2)+ ,(10) ـ


6(, +)3) ـ 6(, +)3) ـ ( ) 8 Fusarium oxysporum

4(, +)4) ـ ـ( 8) ( 61 Fusarium solani

3(, +)5) ـ 5(, +)3) ـ ( ) 61 Fusarium solani

Fusarium acuminatum 44 (7)+ ,(5) ـ (7)+ ,(5) ـ

(8+) (8+) 61 Fusarium avenaceum

ـ( 4) ـ( 4) 8 Fusarium equiseti

ـ( 4) ـ( 4) 8 Fusarium moniliforme

6(, +)7) ـ 3(, +)5) ـ ( ) 61 Fusarium moniliforme

ـ( 4) ـ( 4) 8 Fusarium graminearum

6(, +)7) ـ 6(, +)7) ـ ( ) 61 Fusarium culmorum

ـ( 4) ـ(4) 8 Fusarium chlamydosporum a Total number of tubes in each test.

b Negative or positive sign in front of each number means that the content of the tube has frozen or has no frozen, respectively.

Also, the numbers represent the number of tubes. In cases where at least one tube showed a positive response, the experiment was conducted with more eight or sixteen replicates in order to ensure.

Microorganisms ice nucleation activity So far, few studies have been conducted on the fungi having ice nucleation activity. In this regard, such ice nuclei formation abilities have been identified in some fungal species such as F. tricinctum (Richard, 1996, Tsumuki, 1995), F. avenaceum (Hasegawa, 1994; Pouleur, 1992; Tsumuki , 1995), F. acuminatum (Humphreys, 2001; Pouleur, 1992), F. oxysporum (Richard, 1996) and F. moniliform var. subglutinans (Tsumuki, 1995). Also, Kieft and Ruscetti (1990) reported such activities in mycobiont of Rhizoplaca chrysoleuca at the temperatures above -5

°C.

The application of the tube nucleation test in the present study confirmed that just some of the Fusarium species present ice nucleation activity and other studied genera lack such a feature (Table 1). In this regard, Rhizoctonia solani, Trichoderma virens, Verticillinm dahliae, Aspergillus spp., Alternaria alternata, Alternaria solani Penicillium spp. were found to have no ice nucleation activity which is completely consistent with the findings of Pouleur, (1992). In addition, the results illustrated no ice nucleation activity in Nattrassia spp., Macrophomina phaseolina, Magnoporthe salvinii and Ganmannomyces spp. Consequently, drop freezing tests were conducted on F. avenaceum and F. acuminatum, considering their observed remarkable ice nucleation activity.


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Ice nucleation activity in Fusarium avenaceum Although no statistically significant difference was observed between the suspension concentrations of 1 mg/mL and 2 mg/mL, such differences between other suspension concentrations were significant (see Table 2). This means that the ice nucleation activity of F. avenaceum is enhanced as the suspension concentration (factor (A)) increases, as stated by Lindow et al. (1982). As presented in Fig. 1, the highest ice nucleation activity of the F. avenaceum was observed at the suspension concentration of 5 mg/mL and -7

°C at which all the suspension drops

were frozen.

Table 2. Comparison of the means of the frozen drops of different Fusarium avenaceum suspension concentrations 10 (mg/mL) 5 (mg/mL) 2 (mg/mL) 1 (mg/mL) 0 (mg/mL) (control) Suspension concentration

0.9860*±0.03322 0.9071

*±0.03322 0.8014

*±0.03322 0.7641

*±0.03322 - 0 (mg/mL) (control)

0.2219*±0.03322 0.1430

*±0.03322 0.0373

ns±0.03322 - 0.7641

*±0.03322 1 (mg/mL)

0.1846*±0.03322 0.1057

*±0.03322 - 0.0373

ns±0.03322 0.8014

*±0.03322 2 (mg/mL)

0.0789*±0.03322 - 0.1057

*±0.03322 0.1430

*±0.03322 0.9071

*±0.03322 5 (mg/mL)

- 0.0789*±0.03322 0.1846

*±0.03322 0.2219

*±0.03322 0.9860

*±0.03322 10 (mg/mL)

LSD 5 % = 0.0742 (*) and (ns) are, respectively, significant and insignificant at a probability level of 5 %.

It can be seen in Table 3 that no considerable differences can be observed between the effects of time durations of 5 min and 10 min, 10 min and 15 min, 15 min and 20 min, 20 min and 25 min and 25 min and 30 min on the F. avenaceum ice nucleation activity. However, the differences between the effects of other time durations were significant. In this regard, the maximum difference was observed between the effects of 5 min and 30 min. So, it can be stated that as the exposure time of F. avenaceum to the temperatures below zero prolongs, the freezing rate increases.

Table 3. Comparison of the means of the frozen drops of Fusarium avenaceum suspension in different time durations 30 (min) 25 (min) 20 (min) 15 (min) 10 (min) 5 (min) Time durations

0.2537*±0.03075 0.2078

*±0.03075 0.1651

*±0.03075 0.1120

*±0.03075 0.0524

ns±0.03075 - 5 (min)

0.2013*±0.03075 0.1554

*±0.03075 0.1127

*±0.03075 0.0596

ns±0.03075 - 0.0524

ns±0.03075 10 (min)

0.1417*±0.03075 0.0958

*±0.03075 0.0531

ns±0.03075 - 0.0596

ns±0.03075 0.1120

*±0.03075 15 (min)

0.0886*±0.03075 0.0427

ns±0.03075 - 0.0531

ns±0.03075 0.1127

*±0.03075 0.1651

*±0.03075 20 (min)

0.0459ns

±0.03075 - 0.0427ns

±0.03075 0.0958*±0.03075 0.1554

*±0.03075 0.2078

*±0.03075 25 (min)

- 0.0459ns

±0.03075 0.0886*±0.03075 0.1417

*±0.03075 0.2013

*±0.03075 0.2537

*±0.03075 30 (min)


The ice nucleation activity of F. avenaceum was found to increase with decrease in temperature from 4ـ

°C to ـ

8 °C. However, further decrease in temperature from 8ـ

°C to 9ـ

°C did not enhance the ice nucleation activity,

(Table 4; Fig. 1). Moreover, F. avenaceum subjected to exposure to higher temperatures (23 °C to 90

°C) prior to

cold treatment, indicated that ice nucleation activity increased as temperature decreased. However, such decreases were not significantly different between the temperatures from 50

°C to 23

°C (Table 5). Further, no

significant difference between the effects of temperatures 80 °C and 90

°C was observed (see Fig. 2).

Table 4. Comparison of the means of the frozen drops of Fusarium avenaceum suspension at different temperatures

- 9 (°C) - 8

(°C) - 7

(°C) - 6

(°C) - 5

(°C) - 4

(°C) Temperatures

0.7251*±0.03075 0.6713

*±0.03075 0.5290

*±0.03075 0.4020

*±0.03075 0.1392

*±0.03075 - - 4

(°C)

0.5859*±0.03075 0.5321

*±0.03075 0.3898

*±0.03075 0.2658

*±0.03075 - 0.1392

*±0.03075 - 5

(°C)

0.3231*±0.03075 0.2693

*±0.03075 0.1270

*±0.03075 - 0.2658

*±0.03075 0.4020

*±0.03075 - 6

(°C)

0.1961*±0.03075 0.1423

*±0.03075 - 0.1270

*±0.03075 0.3898

*±0.03075 0.5290

*±0.03075 - 7

(°C)

0.0538ns

±0.03075 - 0.1423*±0.03075 0.2693

*±0.03075 0.5321

*±0.03075 0.6713

*±0.03075 - 8

(°C)

- 0.0538ns

±0.03075 0.1961*±0.03075 0.3231

*±0.03075 0.5859

*±0.03075 0.7251

*±0.03075 - 9

(°C)

LSD 5 % = 0.0902 (*) and (ns) are, respectively, significant and insignificant at a probability level of 5 %


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Table 5. Comparison of the means of the frozen drops of Fusarium avenaceum suspension at relatively high temperatures

90 (°C) 80 (

°C) 70 (

°C) 60 (

°C) 50 (

°C) 40 (

°C) 30 (

°C) 23 (

°C)

Temperatures

0.7060*±0.0355

1 0.5569

*±0.0355

1 0.3776

*±0.035

51 0.2327

*±0.035

51 0.0619

ns±0.035

51 0.0153

ns±0.035

51 0.0075

ns±0.035

51 -

23 (°C)

0.6985*±0.0355

1 0.5494

*±0.0355

1 0.3701

*±0.035

51 0.2252

*±0.035

51 0.0544

ns±0.035

51 0.0078

ns±0.035

51 - 0.0075

ns±0.035

51 30 (

°C)

0.6907*±0.0355

1 0.5416

*±0.0355

1 0.3622

*±0.035

51 0.2173

*±0.035

51 0.0465

ns±0.035

51 - 0.0078

ns±0.035

51 0.0153

ns±0.035

51 40 (

°C)

0.6442*±0.0355

1 0.4951

*±0.0355

1 0.3157

*±0.035

51 0.1708

*±0.035

51 - 0.0465

ns±0.035

51 0.0544

ns±0.035

51 0.0619

ns±0.035

51 50 (

°C)

0.4734*±0.0355

1 0.3243

*±0.0355

1 0.1449

*±0.035

51 - 0.1708

*±0.0355

1 0.2173

*±0.0355

1 0.2252

*±0.0355

1 0.2327

*±0.0355

1 60 (

°C)

0.3285*±0.0355

1 0.1794

*±0.0355

1 - 0.1449

*±0.035

51 0.3157

*±0.0355

1 0.3622

*±0.0355

1 0.3701

*±0.0355

1 0.3776

*±0.0355

1 70 (

°C)

0.0293ns

±0.03551

- 0.1794*±0.035

51 0.3243

*±0.035

51 0.4951

*±0.0355

1 0.5416

*±0.0355

1 0.5494

*±0.0355

1 0.5569

*±0.0355

1 80 (

°C)

- 0.0293ns

±0.03551

0.3285*±0.035

51 0.4734

*±0.035

51 0.6442

*±0.0355

1 0.6907

*±0.0355

1 0.6985

*±0.0355

1 0.7060

*±0.0355

1 90 (

°C)

LSD 5% = 0.0872 (*) and (ns) are, respectively, significant and insignificant at a probability level of 5%.

As presented in Table 6, the results obtained from the variance analysis confirmed that the differences between the effects of the various levels of the studied measures on the Fusarium avenaceum ice nucleation activities are statistically significant. It was also indicated that except the bilateral effect of time duration and heating at higher temperatures prior to cold treatment, the mutual effects between the levels of other measures are significant. Moreover, the trilateral effects between the levels of suspension concentration, time duration and temperature and between levels of suspension concentration, time duration and heating at higher temperatures were not found to be significant. However, the trilateral effect of the time duration, temperature and heating at higher temperatures was significant. Eventually, it was found that the quartet effect of the various levels of studied factors is statistically significant.

Table 6.Variance analysis of the effects of the various factors on the Fusarium avenaceum ice nucleation activity SOV

a DF

b SS

c MS

d F

(A) 4 181.025 452451 1240.926**

(B) 5 21.549 4236 118.173**

(C) 5 199.042 39.808 1091.544**

(D) 7 187.061 412743 732.745**

(A×B) 42 5.531 0.277 7.582**

(A×C) 42 50.711 536.2 69.525**

(A×D) 48 46.879 1.674 45.908**

(B×C) 45 3.345 0.134 3.669**

(B×D) 35 1.408 2224 1.103 ns

(C×D) 35 41.784 1.194 32.735**

(A×B×C) 622 2.949 0.029 0.809ns

(A×B×D) 642 1.354 0.01 0.265ns

(B×C×D) 675 15.386 0.088 2.411**

(A×B×C×D) 6431 661.788 0.548 15.02625**

Error 6431 52.47963 0.03647 -

Total 3477 1012.131 - -

(**) and (ns) are, respectively, significant and insignificant at a probability level of 1 %. CV (Coefficient of Variation) in this test

was equal to 3.65. The Effects of various concentrations of suspension, time, temperature and heat are shown as A, B, C and D, respectively

a Source of variation

b Degrees of freedom

c Sum of squares d

Mean squares


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Ice nucleation activity in Fusarium acuminatum The differences between the effects of the various F. acuminatum suspension concentrations are presented in table 7. Except the difference between the effects of the suspension concentrations of 1 mg/mL and 2 mg/mL of F. acuminatum, the observed differences between other concentrations were found to be significant. Further, as the concentration of F. acuminatum suspension increased, the ability of the ice nuclei formation showed a tangible increase, comparable to that for F. avenaceum. However, the highest ice nucleation activity of the F. acuminatum achieved at the suspension concentration of 10 mg/mL and -7

°C at which all the suspension drops were frozen

(see Figure 1).

Table 7. Comparison of the means of the frozen drops of different Fusarium acuminatum suspension concentrations 10 (mg/mL) 5 (mg/mL) 2 (mg/mL) 1 (mg/mL) 0 (mg/mL) (control) Suspension concentration

0.8419*±0.03441 0.7616

*±0.03441 0.6822

*±0.03441 0.6405

*±0.03441 - 0 (mg/mL) (control )

0.2014*±0.03441 0.1211

*±0.03441 0.0417

ns±0.03441 - 0.6405

*±0.03441 1 (mg/mL)

0.1597*±0.03441 0.0794

*±0.03441 - 0.0417

ns±0.03441 0.6822

*±0.03441 2 (mg/mL)

0.0803*±0.03441 - 0.0794

*±0.03441 0.1211

*±0.03441 0.7616

*±0.03441 5 (mg/mL)

- 0.0803*±0.03441 0.1597

*±0.03441 0.2014

*±0.03441 0.8419

*±0.03441 10 (mg/mL)


In terms of the effects of various time durations on the F. acuminatum ice nucleation activity, it can be stated that there were no significant differences between time durations of 5 min and 10 min, 10 min and 15 min, 15 min and 20 min, 20 min and 25 min and 25 min and 30 min. There were however significant differences between other time durations, as presented in table 8. The results showed the longer the duration of exposure to cold, the more the freezing.

Table 8. Comparison of the means of the frozen drops of Fusarium acuminatum suspension in different time durations 30 (min) 25 (min) 20 (min) 15 (min) 10 (min) 5 (min) Time durations

0.2543*±0.03186 0.1982

*±0.03186 0.1404

*±0.03186 0.0986

*±0.03186 0.0558

ns±0.03186 - 5 (min)

0.1986*±0.03186 0.1424

*±0.03186 0.0846

*±0.03186 0.0429

ns±0.03186 - 0.0558

ns±0.03186 10 (min)

0.1557*±0.03186 0.0996

*±0.03186 0.0418

ns±0.03186 - 0.0429

ns±0.03186 0.0986

*±0.03186 15 (min)

0.1139*±0.03186 0.0578

ns±0.03186 - 0.0418

ns±0.03186 0.0846

*±0.03186 0.1404

*±0.03186 20 (min)

0.0561ns

±0.03186 - 0.0578ns

±0.03186 0.0996*±0.03186 0.1424

*±0.03186 0.1982

*±0.03186 25 (min)

- 0.0561ns

±0.03186 0.1139*±0.03186 0.1557

*±0.03186 0.1986

*±0.03186 0.2543

*±0.03186 30 (min)


As can be seen in Table 9, except the temperatures -8

°C and -9

°C, the effects of other temperatures on the ice

nucleation activity of F. acuminatum were significantly different. Such findings are concordant with the ice nucleation behavior of F. avenaceum, when exposed to the similar temperatures. It was also reflected in the results presented in table 9 that ice nucleation activity showed a significant increase as the temperature decreased from - 4

°C to - 8

°C (also see Fig. 1).

Table 9. Comparison of the means of the frozen drops of Fusarium acuminatum suspension at different temperatures

- 9 (°C) - 8

(°C) - 7

(°C) - 6

(°C) - 5

(°C) - 4

(°C) Temperatures

0.7248*±0.03186 0.6634

*±0.03186 0.5008

*±0.03186 0.3586

*±0.03186 0.0972

*±0.03186 - - 4

(°C)

0.6277*±0.03186 0.5662

*±0.03186 0.4036

*±0.03186 0.2611

*±0.03186 - 0.0972

*±0.03186 - 5

(°C)

0.3666*±0.03186 0.3052

*±0.03186 0.1426

*±0.03186 - 0.2611

*±0.03186 0.3586

*±0.03186 - 6

(°C)

0.2240*±0.03186 0.1626

*±0.03186 - 0.1426

*±0.03186 0.4036

*±0.03186 0.5008

*±0.03186 - 7

(°C)

0.0614ns

±0.03186 - 0.1626*±0.03186 0.3052

*±0.03186 0.5662

*±0.03186 0.6634

*±0.03186 - 8

(°C)

- 0.0614ns

±0.03186 0.2240*±0.03186 0.3666

*±0.03186 0.6277

*±0.03186 0.7248

*±0.03186 - 9

(°C)


As the results presented in Table 10 and Fig. 2 reveal, a slight decrease can be observed in the ice nucleation activity of F. acuminatum when exposed to temperatures between 23

°C to 50

°C prior to prior to cold treatment.

However, there are no significant differences between the effects of other temperatures (23 °C and 30

°C and 23

°C

and 40 °C as well as 30

°C and 40

°C, 40

°C and 50

°C and 30

°C and 50

°C) on the ice nucleation activity of F.

acuminatum. Moreover, although between temperatures 80 °C and 90

°C no significant difference was identified,


525

the differences between other temperatures were found to be statistically significant. Such achievements support the hypothesis that exposure to high temperatures before cold treatment can reduce the ice nucleation activity of F. acuminatum.

Table 10. Comparison of the means of the frozen drops of Fusarium acuminatum suspension at relatively high temperatures

90 (°C) 80 (

°C) 70 (

°C) 60 (

°C) 50 (

°C) 40 (

°C) 30 (

°C) 23 (

°C)

Temperatures

0.6559*±0.0367

8 0.5716

*±0.0367

8 0.3743

*±0.036

78 0.2321

*±0.036

78 0.0785

*±0.0367

8 0.0201

ns±0.036

78 0.0120

ns±0.036

78 - 23 (

°C)

0.6439*±0.0367

8 0.5595

*±0.0367

8 0.3622

*±0.036

78 0.2201

*±0.036

78 0.0664

ns±0.036

78 0.0081

ns±0.036

78 -

0.0120ns

±0.03678

30 (°C)

0.6358*±0.0367

8 0.5514

*±0.0367

8 0.3541

*±0.036

78 0.2120

*±0.036

78 0.0583

ns±0.036

78 -

0.0081ns

±0.03678

0.0201ns

±0.03678

40 (°C)

0.6442*±0.0367

8 0.4951

*±0.0367

8 0.2958

*±0.036

78 0.1536

*±0.036

78 -

0.0583ns

±0.03678

0.0664ns

±0.03678

0.0785*±0.0367

8 50 (

°C)

0.4238*±0.0367

8 0.3395

*±0.0367

8 0.1422

*±0.036

78 -

0.1536*±0.0367

8 0.2120

*±0.0367

8 0.2201

*±0.0367

8 0.2321

*±0.0367

8 60 (

°C)

0.2816*±0.0367

8 0.1973

*±0.0367

8 -

0.1422*±0.036

78 0.2958

*±0.0367

8 0.3541

*±0.0367

8 0.3622

*±0.0367

8 0.3743

*±0.0367

8 70 (

°C)

0.0128ns

±0.03678

- 0.1973

*±0.036

78 0.3395

*±0.036

78 0.4951

*±0.0367

8 0.5514

*±0.0367

8 0.5595

*±0.0367

8 0.5716

*±0.0367

8 80 (

°C)

- 0.0128

ns±0.036

78 0.2816

*±0.036

78 0.4238

*±0.036

78 0.6442

*±0.0367

8 0.6358

*±0.0367

8 0.6439

*±0.0367

8 0.6559

*±0.0367

8 90 (

°C)


Like those for F. avenaceum, the results provided in Table 11 indicate that the effects of various levels of factors (A), (B), (C) and (D) on the ice nucleation activity of F. acuminatum are significantly different. The investigations of the studied factors mutual effects indicated that there are significant differences between the levels of factors (A) and (B), (A) and (C), (A) and (D), (B) and (C) and (C) and (D), while the mutual effects of various levels of (B) and (D) were not found to be significant. Findings also showed that there is a significant difference between the effects of levels of factors (B), (C) and (D), while no significant difference was observed between the effects of levels of (A), (B) and (C) and also between the effects of levels of (A), (B) and (D), similar to those achieved for F. avenaceum.

Table 11. Variance analysis of the effects of various factors on the Fusarium acuminatum ice nucleation activity SOV

a DF

b SS

c MS

d F

(A) 4 125.284 31.321 498.031**

(B) 5 20.365 4.073 64.765**

(C) 5 198.225 39.645 630.39**

(D) 7 168.004 24.001 381.629**

(A×B) 42 6.84 0.342 5.438**

(A×C) 42 51.229 2.561 40.73**

(A×D) 48 41.962 1.499 23.83**

(B×C) 45 6.123 0.245 3.894**

(B×D) 35 3.053 0.087 1.387ns

(C×D) 35 59.577 1.702 27.066**

(A×B×C) 622 8.554 0.086 1.36ns

(A×B×D) 642 2.562 0.018 0.291ns

(B×C×D) 675 15.491 0.089 1.408**

(A×B×C×D) 6431 741.395 0.515 0.462ns

Error 6431 90.49822 0.06289 -

Total 3477 1539.162 - -

(**) and (ns) are, respectively, significant and insignificant at a probability level of 1 %. CV (Coefficient of Variation) in this test

was equal to 5.24. The effects of various concentrations of suspension, time, temperature and heat are shown as A, B, C and D, respectively.

a Source of variation

b Degrees of freedom

c Sum of squares d

Mean squares


526

Comparison of the Fusarium avenaceum and Fusarium acuminatum INAs According to the results presented in Fig. 1, the highest temperature at which F. avenaceum suspension began to freeze was found to be -4

°C at a concentration of 2 mg/mL, while F. acuminatum at the same concentration

showed ice nucleation activity at a temperature of -5 °C. In this regard, Pouleur et al. (Kieft and Ruscetti, 1990)

reported the ice nucleation activity of F. avenaceum suspension at a maximum temperature of -2.5 °C. Also

Tsumuki et al. (Knopf, 2011) reported the ice nucleation activity of F. avenaceum suspension at a temperature of -6 °C. further, In a study conducted on cloud seeding by using fungal microorganisms, ice nucleation activity of F. avenaceum was reported at a temperature of -6

°C and it was stated that the microorganisms having ice nucleation

activity in rain clouds can play an active role in controlling the physical properties of atmospheric water (Zhang, 2010).

Figure 1. Comparison of the effects of various temperatures on the rate of the ice nucleation activity of Fusarium avenaceum () and Fusarium acuminatum ( ) as the percentage of the frozen drops after exposure to different suspension concentrations (a:

1 mg/mL, b: 2 mg/mL, c: 5 mg/mL and d: 10 mg/mL) for 30 min with three replicates

From the Fig. 1, it can also be stated that F. avenaceum has a stronger ice nucleation activity than F. acuminatum. In addition, it is clear that as the temperature decreases, the difference between the ice nucleation activities of them reduces. This means that at temperatures lower than -7

°C, both studied species show an intense

ice nucleation activity and almost all of their suspension drops (in drop freezing test) freezes. Moreover, the ice nucleation activity intensifies as the concentration of suspension increases. According to the results of the drop freezing test, 100 % of drops of fungal suspension concentrations of 2 mg/mL, 5 mg/mL and 10 mg/mL at temperatures of -8

°C and -9

°C were frozen. Additionally, all drops of different suspensions were frozen at a

concentration of 10 mg/mL at a temperature of -7 °C. 100 % of drops of F. avenaceum suspension were also frozen

at a concentration of 5 mg/mL. At a concentration of 1 mg/mL, none of the fungal suspension showed ice nucleation activity at a temperature of 4ـ

°C and freezing rate was observed equal to 0. No ice nucleation activity

was observed in F. acuminatum at a concentration of 2 mg/mL and at a temperature of -4 °C, while 7 % and 20 %

of drops were frozen at concentrations of 5 mg/mL and 10 mg/mL, respectively. At a concentration of 2 mg/ml and at a temperature of -4

°C, about 7 % of F. avenaceum suspension drops, whereas at concentrations of 5 mg/ml and

10 mg/ml at the same temperature, about 20 % and 30 % of drops were frozen, respectively. Fig. 2 presents the ice nucleation behavior of the F. avenaceum and F. acuminatum when their suspension concentrations of 1 mg/mL were exposed to temperatures from 23 °C to 90 °C for 10 min, before rapid cooling to 20 °C. In this regard, the results obtained from drop freezing test revealed that the ability of ice nucleation of fungi reduces by increased heat. This reduction is more significant at temperatures above 60

°C. Such findings are

concordant with Pouleur et al. (Kieft and Ruscetti, 1990) which reported that fungal ice nucleation activity reduces after being exposed to temperatures above 60

°C for 10 min.

Based on the results of the present study and similar studies, ice nucleation process applications such as searching for finding the sources of biological ice nuclei for cloud seeding to produce artificial rain and snow, given the drought and lack of rainfall in recent years, can be taken into account. Additionally, the features of ice core can be useful in controlling biological insects and storage pests. Other applications of this process include the biotechnology of producing negative mutants of ice core and preventing frostbite and hypothermia of plants, the use of ice nucleation features in food industry, freezing the food tissue and other commercial uses.


527

Figure 2. Comparison of the effects of the heating at 23 °C, 30 °C, 40 °C, 50 °C, 60 °C, 70 °C, 80 °C and 90 °C for 10 min prior to cold treatments on the Fusarium avenaceum ( )and Fusarium acuminatum ( ) ice nucleation activity. The suspension concentrations were 1 mg/mL and the freezing temperatures were recorded based on the freezing of 50 % of the drops with

three replicates.

CONCLUSION

Today, a large number of particles with biological and non-biological origin have been identified that could be used as a core for ice formation, based on supercooling process, in various applications, making possible the beginning of water freezing at higher temperatures. On the other hand, this feature in INA microorganisms can bear side effects such as cold stress in plants and general disorder in plants function. It also may lead to economic and ecological damages through causing damage or destruction to plants. The results related to 39 isolated and identified isolates showed that only some of Fusarium species present the ice nucleation activity and other studied species lacked this feature. The results obtained from drop freezing test on Fusarium avenaceum and Fusarium acuminatum species, which showed significant ice nucleation activity, indicate that these species have some differences from each other in terms of the effect of suspension concentration, temperature, time duration and heating at higher temperatures variables. In this regard, the results showed that F. avenaceum has a stronger ice nucleation activity than F. acuminatum. In addition, it seems that as the temperature decreases, the differences between ice nucleation activities in these microorganisms are reduced. Additionally, this feature increases as the concentration of suspensions is increased. Further, the experiments revealed that increased time duration (from 5 min to 30 min) of exposure to subـzero temperatures (from 4ـ

°C to 9ـ

°C) can caused an increase in ice nucleation activity of suspensions of F. avenaceum and F. acuminatum species. The results also showed that the ice nucleation ability of fungi decreases when they are exposed to high above-zero temperatures.

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Investigation and comparison of the ice nucleation activity in Fusarium avenaceum and Fusarium...

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