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The role of onion-associated fungi in bulb mite infestation and damage to onion seedlings Tal Ofek Shira Gal Moshe Inbar Sara Lebiush-Mordechai Leah Tsror Eric Palevsky Received: 10 April 2013 / Accepted: 1 November 2013 / Published online: 16 November 2013 Ó Springer Science+Business Media Dordrecht 2013 Abstract In Israel Rhizoglyphus robini is considered to be a pest in its own right, even though the mite is usually found in association with fungal pathogens. Plant protection recommendations are therefore to treat germinating onions seedlings, clearly a crucial phase in crop production, when mites are discovered. The aim of this study was to determine the role of fungi in bulb mite infestation and damage to germinating onion seedlings. Accordingly we (1) evaluated the effect of the mite on onion seedling germination and survival without fungi, (2) compared the attraction of the mite to species and isolates of various fungi, (3) assessed the effect of a relatively non-pathogenic isolate of Fusarium oxysporum on mite fecundity, and (4) determined the effects of the mite and of F. oxysporum separately and together, on onion seedling germination and sprout development. A significant reduction of seedling survival was recorded only in the 1,000 mites/pot treatment, after 4 weeks. Mites were attracted to 6 out of 7 collected fungi isolates. Mite fecundity on onion sprouts infested with F. oxysporum was higher than on non-infested sprouts. Survival of seedlings was affected by mites, fungi, and their combination. Sprouts on Petri dishes after 5 days were significantly longer in the control and mite treatments than both fungi treatments. During the 5-day experiment more mites were always found on the fungi-infected sprouts than on the non-infected sprouts. Future research using suppressive soils to suppress soil pathogens and subsequent mite damage is proposed. Keywords Rhizoglyphus robini Á Fusarium oxysporum Á Weakly-pathogenic fungi Á Onion Á Mite–fungal interaction T. Ofek Á S. Gal Á E. Palevsky (&) Department of Entomology, Institute of PlantProtection, Newe-Ya’ar Research Center, Agricultural Research Organization (ARO), Ramat Yishay, Israel e-mail: [email protected] T. Ofek Á M. Inbar Department of Evolutionary and Environmental Biology, University of Haifa, Haifa, Israel S. Lebiush-Mordechai Á L. Tsror Department of Plant Pathology and Weed Research, Gilat Research Center, Institute of Plant Protection, Agricultural Research Organization (ARO), MP Negev, Israel 123 Exp Appl Acarol (2014) 62:437–448 DOI 10.1007/s10493-013-9750-2

The role of onion-associated fungi in bulb mite infestation and damage to onion seedlings

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Page 1: The role of onion-associated fungi in bulb mite infestation and damage to onion seedlings

The role of onion-associated fungi in bulb mite infestationand damage to onion seedlings

Tal Ofek • Shira Gal • Moshe Inbar • Sara Lebiush-Mordechai •

Leah Tsror • Eric Palevsky

Received: 10 April 2013 / Accepted: 1 November 2013 / Published online: 16 November 2013� Springer Science+Business Media Dordrecht 2013

Abstract In Israel Rhizoglyphus robini is considered to be a pest in its own right, even

though the mite is usually found in association with fungal pathogens. Plant protection

recommendations are therefore to treat germinating onions seedlings, clearly a crucial phase

in crop production, when mites are discovered. The aim of this study was to determine the role

of fungi in bulb mite infestation and damage to germinating onion seedlings. Accordingly we

(1) evaluated the effect of the mite on onion seedling germination and survival without fungi,

(2) compared the attraction of the mite to species and isolates of various fungi, (3) assessed the

effect of a relatively non-pathogenic isolate of Fusarium oxysporum on mite fecundity, and

(4) determined the effects of the mite and of F. oxysporum separately and together, on onion

seedling germination and sprout development. A significant reduction of seedling survival

was recorded only in the 1,000 mites/pot treatment, after 4 weeks. Mites were attracted to 6

out of 7 collected fungi isolates. Mite fecundity on onion sprouts infested with F. oxysporum

was higher than on non-infested sprouts. Survival of seedlings was affected by mites, fungi,

and their combination. Sprouts on Petri dishes after 5 days were significantly longer in the

control and mite treatments than both fungi treatments. During the 5-day experiment more

mites were always found on the fungi-infected sprouts than on the non-infected sprouts.

Future research using suppressive soils to suppress soil pathogens and subsequent mite

damage is proposed.

Keywords Rhizoglyphus robini � Fusarium oxysporum � Weakly-pathogenic

fungi � Onion � Mite–fungal interaction

T. Ofek � S. Gal � E. Palevsky (&)Department of Entomology, Institute of PlantProtection, Newe-Ya’ar Research Center, AgriculturalResearch Organization (ARO), Ramat Yishay, Israele-mail: [email protected]

T. Ofek � M. InbarDepartment of Evolutionary and Environmental Biology, University of Haifa, Haifa, Israel

S. Lebiush-Mordechai � L. TsrorDepartment of Plant Pathology and Weed Research, Gilat Research Center, Institute of PlantProtection, Agricultural Research Organization (ARO), MP Negev, Israel

123

Exp Appl Acarol (2014) 62:437–448DOI 10.1007/s10493-013-9750-2

Page 2: The role of onion-associated fungi in bulb mite infestation and damage to onion seedlings

Introduction

The bulb mite Rhizoglyphus robini Claparede (Astigmata: Acaridae) is a pest of flower

bulbs, vegetables and cereals (Diaz et al. 2000). In Israel it attacks onion (Allium cepa),

garlic (Allium sativum), lily (Lilium longiflorum) and ruscus (Danae racemosa) (Ben-

David et al. 2005). Damage has been attributed to the mite itself (Gerson et al. 1985) and to

the combined effects of the mite and fungal pathogens infecting various crops, such as lily

(Ascerno et al. 1981; Czajkowska 1995), Oriental onion (Allium chinense) (Okabe and

Amano 1991), and wheat (Armitage and George 1986). Bulb mite population growth on

rakkyo in the presence of Fusarium oxysporum f. sp. allii was much faster compared with

mechanically injured or healthy bulbs (Okabe and Amano 1991) and the mite was attracted

to alcohols secreted by this fungus, as it was to infested bulbs (Okabe and Amano 1990).

Nesvorna et al. (2012) found that Fusarium spp. sustained the population growth of

Tyrophagus putrescentiae (Schrank) (Acaridae). Using fecundity levels of Tyrophagus

similis Volgin as an indicator for diet suitability, Kasuga and Honda (2006) reported that

organic fertilizer rape-seed meal, vegetables and the fungi F. oxysporum, Pythium

aphanidermatum and Rhizoctonia solani were suitable diets.

At this point we may question whether the mites are primary or secondary pests,

arriving after the establishment of the fungal pathogens. In Israeli garlic and onion fields

the bulb mite is treated as a pest in its own right, even though it is always found in

association with fungal pathogens (Nabil Omari, field crops extension agent, personal

communication).

In a separate study we determined the pathogenicity of species and isolates of fungi,

isolated from onions and lilies in association with the bulb mite in Israel, in an environment

controlled greenhouse. Seedling survival was recorded after 40 days. From the three strains

of F. oxysporum evaluated, one strain was relatively non-pathogenic and did not differ

from the control (Lebiush-Mordechai et al. 2013).

The aim of the present study, in contrast to previous studies on mature plant material

(Okabe and Amano 1991; Ben-David et al. 2005), was to determine the role of fungi in

bulb mite infestation and damage to germinating onion seedlings, clearly a critical stage in

plant development. More specifically we (1) determined the effect of the mite without the

addition of fungi, (2) assessed attraction of the mite to species and isolates of fungi, (3)

evaluated the suitability of onion seedlings, infested or non-infested with a relatively non-

pathogenic isolate of F. oxysporum, as a food source for the mite, (4) separately and

together recorded the effects of the mite and the non-pathogenic fungus on onion seedling

germination and sprout development. It was important to use this relatively non-pathogenic

strain of F. oxysporum because a pathogenic strain would have killed the onion seedlings

irrespective of the bulb mites.

Materials and methods

Rhizoglyphus robini was collected in 2004 from Lily bulbs grown in greenhouses on the

coastal plain in Israel and has since been reared in the lab at the Newe-Ya’ar Research

Center, Agricultural Research Organization (ARO), Israel. Mites were maintained in

culture for experiments that were conducted for 2 years thereafter. Each rearing unit

consisted of a Petri dish padded with wet filter paper and began with 10 females and a tea

spoon of fresh ground peanuts. Arenas were setup weekly, kept in a dark chamber at 25 �C

and discarded after 6 weeks.

438 Exp Appl Acarol (2014) 62:437–448

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Effect of bulb mite on onion seedling survival

The effect of mite densities on onion seedling germination and survival was assessed in

250 ml pots, sown with 16 onion seeds CV ‘Ada 781’, in a sterilized soil mix of 70 % peat

and 30 % perlite (Shacham Givat Ada, Israel) containing a slow release fertilizer 0.06 g/l

Osmocote� (Scotts Miracle-Gro, USA). Each pot was glued to a plastic container cover

(Meitav, Plastic and Packaging Products, Holon, Israel), bottom side up. The lip of the

cover served as a moat filled with castor oil, to prevent mite migration from one pot to

another. Both pots and plastic container cover were pierced at the bottom for drainage. Pots

were irrigated every 3 days with 100 ml distilled water. To quantitatively inoculate the

pots with bulb mites we used the following procedure: Rearing arenas with mites and

peanuts were washed under tap water through a coarse sieve 710 lm and fine sieve 180 lm

(Ari J. Levy—Laboratory Equipment, Petach Tikva, Israel). Washed mites collected in the

180 lm sieve were back washed with distilled water, and mite numbers were estimated by

10 counts of a 1 ml volume suspension. Pots were then inoculated with 0, 10, 100 and

1,000 mites (all motile stages) in 100 ml of distilled water, replicated 6 times. The

experiment was conducted in a standard refrigerator size incubator (Dahan Technologies,

Israel) at 11:13 L:D, day 28 ± 2 �C, night 17 ± 2 �C and 68 ± 9 % RH. These conditions

are typical of the Israeli early autumn, when R. robini infestations often occur on onion

seedlings in open fields. Seedling survival were recorded over the 4 weeks of the exper-

iment and calculated as the percentage of seedlings that germinated and survived from the

number of seeds planted. Seedling survival was analyzed with repeated measures ANOVA,

means separated by Tukey honestly significant differences (HSD) (JMP� 10.0.0, SAS

Institute). At the end of the experiment mites were extracted by using Berlese funnels and

counted. Mite counts were subjected to log (x ? 1) transformation to attain normality.

Data were analyzed by ANOVA, means separated by Tukey HSD.

Attraction of bulb mite to fungal species and isolates

Three isolates of F. oxysporum, Fusarium proliferatum, Pythium oligandrum, bi-nuclei

Rhizoctonia AG-A, and Verticillium dahliae were collected in Israel in 2005, cultured and

maintained at the Gilat Research Center, ARO (Table 1) (Lebiush-Mordechai et al. 2013).

To prepare potato dextrose agar (PDA DifcoTM 500 g) infested sections for the experi-

ments described below, and for maintenance of fungal colonies, PDA squares of 10 mm

were cut from infested Petri dishes kept at 4 �C and transferred to new Petri dishes

(90 mm dia) at 25 �C for 15 days, by which time the dishes were fully covered with

mycelium.

Experiments to determine the attraction of mites to the collected fungi were conducted

in disposable 50 mm Petri dishes on wet filter paper. Four PDA sections (5 mm dia) were

placed adjacent to the rim of the Petri dish at equal distances from each other and from the

center, two colonized with one fungal species/isolate (infested as above) and two non-

colonized (controls), replicated six times for each fungus species/isolate. The experiment

was conducted at 25� ± 1 �C, ten females of R. robini were placed at the center of the

plate and the number of mites found on inoculated and control sections were recorded

every hour for 4 h. The attraction of each fungus species/isolate, as compared to its control,

was analyzed after 4 h with the nonparametric paired sample Wilcoxon signed rank test

(SPSS, version 20).

Exp Appl Acarol (2014) 62:437–448 439

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Page 4: The role of onion-associated fungi in bulb mite infestation and damage to onion seedlings

The effect of a non-pathogenic fungus on the fecundity of bulb mite

The effect of F. oxysporum isolate III, which was relatively non-pathogenic to onion

seedlings in a potted plant trial (Lebiush-Mordechai et al. 2013), on R. robini fecundity,

was evaluated on onion sprouts. An even-aged cohort of young ovipositing mites was

obtained by transferring 20 adult females to a new rearing arena (as above) to lay eggs for

24 h. The adult mites were then removed, leaving the eggs that were laid to develop.

Fifteen days later (Gerson et al. 1983), 100 female and 100 male adults were transferred to

20 wells, 16 mm diameter (Greiner Bio One, Germany) lined with wet filter paper without

food, each well containing five males and five females, to ensure mating. Before placing

the mites on onion sprouts, with or without the fungus, mites were starved for 5 days, thus

allowing for a more accurate evaluation of the effect of the fungus on the mite’s fecundity.

To prevent dehydration during the 5 day starvation period, 60 ll of distilled water was

dripped every 48 h into each well. To inoculate onion seedlings with the relatively non-

pathogenic fungus we used the following procedure. Thirty seedlings were allowed to

germinate in a 90 mm Petri dish on wet filter paper. After 7 days 15 seedlings were

transferred to a Petri dish with PDA, inoculated with F. oxysporum isolate III (as described

above for the attraction of R. robini to fungal species and isolates), leaving the remaining

non-inoculated 15 seedlings in the dish on wet filter paper. After 3 additional days, each of

the 30 sprouts (15 with and 15 without the fungus) was transferred to a 90 mm Petri dish

padded with wet filter paper, and one starved mated female was added with a fine brush.

Five days later the eggs per dish were counted and the effect of the fungus on mite

fecundity was determined with a non-paired t test.

The effect of fungal–mite interactions on onion seedling and sprout development

The effects on onion seedling and sprout development of the mite, the fungus

F. oxysporum isolate III and their combination, versus a non-treated control, were studied in

two sets of experiments, in pots and Petri dishes, differing in both spatial and temporal scales.

Similar to the experiment determining mite effect on onion seedling survival, 32 pots of

360 ml were sown, each with 12 onion seeds, allowing for 8 replicates of the four treat-

ment combinations (mites only, fungus only, mites and fungus, and the non-treated con-

trol). To 16 pots we applied 4 ml of a conidial suspension of the F. oxysporum isolate III,

containing ca. 4 9 105 colony forming units. To 8 pots inoculated with the fungus and 8

without, we added 100 females/pot. The eight remaining pots were neither infested with

Table 1 Collection sites and plant hosts for three isolates of Fusarium oxysporum (referred to as isolates I,II and III), Fusarium proliferatum, Pythium oligandrum, bi-nuclei Rhizoctonia AG-A, and Verticilliumdahliae collected in Israel, 2005

Fungi species Isolate Isolate color Plant Site

Fusarium oxysporum I White Onion Beit She’an

Fusarium oxysporum II Purple Lilly Beerotayim

Fusarium oxysporum III Dark purple Onion Beit She’an

Fusarium proliferatum White Onion Beit She’an

Pythium oligandrum Lilly Kfar ha-Ro’e

bi-nuclei Rhizoctonia AG-A Lilly Kfar ha-Ro’e

Verticillium dahliae Potato Northern Negev

440 Exp Appl Acarol (2014) 62:437–448

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Page 5: The role of onion-associated fungi in bulb mite infestation and damage to onion seedlings

fungi nor inoculated with mites. Pots were kept in an incubator (as above) at 11:13 L:D,

day 28 ± 3 �C, night 17 ± 3 �C and 75 ± 15 % RH conditions. Seedling survival was

recorded after 7 weeks; mites were extracted to ethanol 70 % using Berlese funnels and

counted under a stereo microscope. Data were analyzed with ANOVA, followed by means

separation with Tukey HSD (JMP � 10.0.0, SAS Institute).

The Petri dish seedling experiment was conducted with the same four treatment combi-

nations, replicated 10 times. Inoculation of 20 sprouts, one sprout per Petri dish, with the

F. oxysporum isolate III, was performed as above for the effect of the fungus on mite

fecundity. Twenty adult females per Petri dish were added to 20 Petri dishes 3 days post-

inoculation, 10 with the fungus and 10 without. The experiment was conducted in a room with

natural sunlight at 25 ± 1 �C and monitored for 5 days. All dishes were photographed daily

with a digital camera, using the macro mode (Nikon 5600 Coolpix). Sprout root length as an

indicator of sprout development was measured from the digital images (Olympus Soft

Imaging System, Germany), and the number of mites per sprout was recorded. Data were

analyzed with repeated measures ANOVA (JMP � 10.0.0, SAS Institute).

Results

Effect of Rhizoglyphus robini on onion seedling survival

The effect of R. robini on onion seedling survival was clearly evident at the highest infestation

level (1,000 mites/pot applied at the beginning of the experiment), 1 week post-sowing, the

trend persisting till the end of the experiment (Fig. 1). Onion seedling survival was signifi-

cantly reduced by mite infestation and time (Table 2). The highest mite treatment had lower

seedling survival rates compared to the two lower levels (100 and 10 mites/pot), which were

similar to the non-treated control (Fig. 1). Mite numbers extracted from the pots at the end of

the experiment were significantly affected by treatment (Table 3), populations being similar

at the two highest infestation rates of 1,000 and 100 mites/pot, which differed from the 10

mites/pot, the latter being different from the non-treated control.

Attraction of Rhizoglyphus robini to fungal species and isolates

Generally the mites were attracted to infested PDA sections. More mites were recorded on

infested sections than on non-infested sections after 4 h, compared to the observations

recorded in the first hour, except for 2-nuclei Rhizoctonia AG-A (Fig. 2). It is interesting to

note that the attraction of R. robini to V. dahliae, a pathogen of many hosts but not of

onions, was even more significant than mite attraction to the pathogenic species

F. oxysporum isolate I (Table 4) found on rotting onions (Table 1). In contrast bi-nuclei

Rhizoctonia AG-A, a species pathogenic to onion, was not attractive to the mite.

The effect of a non-pathogenic fungus on Rhizoglyphus robini fecundity

Mite fecundity on onion sprouts infested with F. oxysporum isolate III was significantly

higher than on non-infested onions. Mean number of eggs/day/dish (for five females) was

39.7 on infested plants compared to 6.6 eggs on non-infested plants (F1,28 = 34.3,

P \ 0.001). It should be noted that the mites were starved for 5 days prior to this evalu-

ation, which may explain the overall low fecundity levels in both treatments.

Exp Appl Acarol (2014) 62:437–448 441

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The effect of fungal–mite interactions on onion seedling and sprout development

Seedling survival was significantly reduced by mites, fungi, time and their interactions,

except for the interaction between mites and time (Table 5). Seedling survival deteriorated

by week 9 in all treatments (mites, fungi, mites and fungi) and was substantially inferior to

the non-treated control (Fig. 3). The treatments fungi and fungi with mites had no effect on

the number of mites extracted at the end of the experiment (Table 6). When only the mite-

infested treatments were considered, significantly more mites were extracted on the fungi-

infested treatment (F1,13 = 4.96, P = 0.044). Still, mite levels after 11 weeks, even in the

fungi-infested pots, did not exceed the initial infestation level (100 mites/pot), indicating

that conditions were not optimal for their development.

Sprout length was significantly affected by mites, fungi, time and their interactions with

time, but unaffected by the interaction between mite and fungi (Table 7). The effect of

Fig. 1 The effects of 0, 10, 100 and 1,000 Rhizoglyphus robini per pot on onion seedling germination andsurvival during 4 weeks. Data were analyzed with repeated measures ANOVA, means separated by TukeyHSD. Different capital letters indicate a significant difference between treatments

Table 2 Repeated measures analysis for the effects of Rhizoglyphus robini (mites), time and their inter-action on seedling survival during 4 weeks in pots infested with 0, 10, 100 and 1,000 mites

DF DFDen F P

Mites 3 19 18.45 0.0001

Time 3 60 46.45 0.0001

Mites 9 time 9 60 0.71 0.70

Block 1 19 2.45 0.13

Table 3 ANOVA for the meannumber of Rhizoglyphus robini/pot extracted after 4 weeks(F3,20 = 49.54, P \ 0.0001)

Different lowercase lettersindicate a significant differencebetween treatments (a = 0.05)

Initial treatmentMites/pot

ExtractedMites/pot

0 1 a

10 9 b

100 46 c

1,000 156 c

442 Exp Appl Acarol (2014) 62:437–448

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Page 7: The role of onion-associated fungi in bulb mite infestation and damage to onion seedlings

fungi and time on the reduction of sprout elongation were far more pronounced than that of

mites. Sprout elongation of the non-treated control and of the mites-only treatment were

similar, both significantly longer than the two fungal treatments (with and without mites),

the latter two being similar (Fig. 4a). Sprout length increased in the control and mites

treatments from day 1, whereas sprout growth was negligible in the fungus and the fungus

and mite treatments. Mycelia were clearly visible on the fungi-infested sprouts without

Fig. 2 Attraction of Rhizoglyphus robini to infested sections with the following fungal species and isolates,collected in Israel in 2005: three isolates of Fusarium oxysporum (referred to as isolates I, II and III),Fusarium proliferatum, Pythium oligandrum, bi-nuclei Rhizoctonia AG-A, and Verticillium dahliae

Table 4 Nonparametric paired sample Wilcoxon signed rank test analysis for the effects of three isolates ofFusarium oxysporum (referred to as isolates I, II and III), F. proliferatum, Pythium oligandrum, 2-nucleiRhizoctonia AG-A, and Verticillium dahliae (fungi) on Rhizoglyphus robini attraction to infested and non-infested agar plugs (n = 6)

Z P

Fusarium oxysporum-I 2.226 0.026

Fusarium oxysporum-II 2.207 0.027

Fusarium oxysporum-III 2.226 0.026

Fusarium proliferatum 1.997 0.046

Pythium oligandrum 1.997 0.046

2-nuclei Rhizoctonia AG-A 0.135 0.89

Verticillium dahliae 2.060 0.039

Exp Appl Acarol (2014) 62:437–448 443

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mites at the end of the experiment, whereas sprouts with mites and fungi were clean,

indicating that the mites had fed on them. Mite attraction to onion sprouts was affected by

F. oxysporum isolate III (fungi), by time, but not by their interaction (Table 8). During the

5 day experiment more mites were always found on the sprouts infested with F. oxysporum

(Fig. 4b). Also noteworthy is that the mites gathered around and fed on the onion sprouts

infested with the fungus, polishing off the mycelium within 24 h, but showed no attraction

or feeding on the non-infested onion sprout.

Table 5 Repeated measures analysis for the effects of Rhizoglyphus robini (mites), Fusarium oxysporumisolate III (fungi), time and their interactions on onion seedling survival in pots over 11 weeks

DF DFDen F P

Mites 1 25 10.97 0.0028

Fungi 1 25 11.25 0.0025

Time 9 252 147.39 \0.0001

Fungi 9 mites 1 25 4.72 0.040

Fungi 9 time 9 252 3.01 0.002

Mites 9 time 9 252 0.95 0.48

Mites 9 fungi 9 time 9 252 1.96 0.045

Block 3 25 17.34 \0.0001

Table 6 ANOVA for the effects of Rhizoglyphus robini (mites), Fusarium oxysporum isolate III (fungi)and their interactions on mites extracted from potted seedlings after 11 weeks

DF F P

Fungi 1 1.14 0.30

Mites 1 43.68 \0.0001

Fungi 9 mites 1 3.70 0.066

Block 3 0.10 0.96

Fig. 3 The effects of Rhizoglyphus robini, Fusarium oxysporum isolate III, and mites and fungi, on onionseedling survival in pots over 11 weeks. Data were analyzed with repeated measures ANOVA, meansseparated by Tukey HSD. Different capital letters indicate a significant difference between treatments

444 Exp Appl Acarol (2014) 62:437–448

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Table 7 Repeated measures analysis for the effects of Rhizoglyphus robini (mites), Fusarium oxysporumisolate III (fungi), time and their interactions on sprout elongation for days 1–5

DF DFDen F P

Mites 1 36 4.59 0.039

Fungi 1 36 54.89 \0.0001

Time 4 144 93.26 \0.0001

Fungi 9 mites 1 36 2.29 0.14

Mites 9 time 4 144 6.12 0.0001

Fungi 9 time 4 144 64.05 \0.0001

Mites 9 fungi 9 time 4 144 3.80 0.0057

Table 8 Repeated measures analysis for the effects of Fusarium oxysporum isolate III (fungi), time andtheir interactions on mite attraction to sprouts with and without fungi for days 1–5

DF DFDen F P

Fungi 1 88 123.58 \0.0001

Time 4 88 13.45 \0.0001

Fungi 9 time 4 88 0.53 0.72

Fig. 4 a The effects of Rhizoglyphus robini, Fusarium oxysporum isolate III, and mites and fungi, on sproutelongation for days 1–5. b The effect of F. oxysporum isolate III on R. robini attraction to onion sprouts.Data were analyzed with repeated measures ANOVA, means separated by Tukey HSD. Different capitalletters indicate a significant difference between treatments

Exp Appl Acarol (2014) 62:437–448 445

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Discussion

Our objective was to evaluate the effects of fungi on bulb mite attraction, infestation and

damage to onion seedlings. First we found that onion seedlings survival was only affected

by a very high level (1,000 mites per pot) of mite infestation. Ben-David et al. (2005)

during the winter in an unheated greenhouse in Israel, evaluated the effect of R. robini on

onion and garlic, starting from garlic cloves and onion bulblets (small bulbs), one bulb per

pot. No effect on bulb weight in either host, even at the highest initial infestation

(1,250 mites/bulb in 360 ml pots) was observed in that study. The onion seeds used in the

present study were apparently far more susceptible to mite damage than the bulblets used

by Ben-David et al. (2005). Mites attacking germinating onion seeds feed on the rootlets

and stem, causing substantial damage, whereas mites feeding on bulblets eat away at the

external layers of the bulb, hardly affecting its establishment. Furthermore, in the present

study the experiment was conducted in an incubator at temperatures simulating early

autumn conditions (September–October), which were higher than the ambient winter

temperatures of the study reported by Ben-David et al. (2005), initiated in December.

In the present study the population of the mites, extracted from the soil at the end of the

experiment, especially in the highest initial infestation rate (1,000 mites/pot), dwindled

over the 4-week period instead of escalating and 80 % of the onion seedlings still survived.

Taken together, evidently an indication that onion seedling is not an ideal food source for

the bulb mite. Similarly mite populations extracted from onion bulblets by Ben-David et al.

(2005) were lower than initial infestation rates. But mite populations in the soil were more

than an order of magnitude higher at the low rate and similar in the high rate. One major

difference between these studies is that Ben-David et al. (2005) dispensed the mite sus-

pension on a paper towel on which the bulblet was set and then covered it with soil,

whereas in the present study the mite suspension was added to the soil. A plausible

explanation can be found in Wooddy and Fashing (1993), who showed that paper can serve

as an excellent substrate for the growth of fungal mycelia, which evidently supported the

high populations reported by Ben-David et al. (2005).

Mite fecundity was 6 times higher on onion sprouts infested with F. oxysporum isolate

III, compared with non-infested onions. Similarly Kasuga and Honda (2006) found the

mean fecundity of T. similis to be 4 times higher on F. oxysporum than on Welsh onion

Allium fistulosum, the latter thus being considered a poor food for T. similis. Additional

evidence attesting the suitability of F. oxysporum for mite oviposition was reported by

Czajkowska (1995), where F. oxysporum f. sp. tulipae supported high fecundity levels of

R. echinopus (Fumouze and Robin), collected from lily.

In our potted plant trial, all treatments (mites, fungi, mites with fungi) were similar after

11 weeks, and differed from the control. In contrast, in the short range experiment on

sprouts in Petri dishes, sprout elongation in the fungi treatments with or without mites

came to a halt on the first day, whereas sprout elongation continued over the 5 day

experiment, in the control and mites-only treatments. Although this fungus was non-

pathogenic when compared to a non-treated control and other F. oxysporum isolates in a

potted plant experiment conducted in a greenhouse (Lebiush-Mordechai et al. 2013), it

strongly affected sprout growth. We assume that the mode of fungus infestation used in the

present study (i.e. germinating the sprouts on an infested Petri dish) allowed for an

overdose of inoculum which essentially stopped sprout elongation at day 1. While the short

term Petri dish bioassay is even more remote from field conditions than the potted plant

experiment, especially with respect to the method of fungal infestation, it was instrumental

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in demonstrating that the mites were not attracted to non-infested sprouts and that the mites

actively fed on the mycelium covering the sprouts.

We, as did others, demonstrated that the interaction between the mite and fungi is

expressed by the mites’ attraction to the latter (Okabe and Amano 1990), the mites’ feeding

on mycelium (Nesvorna et al. 2012; Wooddy and Fashing 1993), and the attraction to and

feeding on plant tissues infected with fungi (Okabe and Amano 1991). In addition we

showed in short range experiments that onion sprouts which are uninfected by fungi are not

attacked by R. robini. Evidently the effect of mites alone on plant fitness parameters is

density and time dependent and is enhanced in the presence of fungi. The ability of

R. robini to digest fungi has recently been attributed to chitinase-producing symbiotic

bacteria (Zindel et al. 2013). A similar process was demonstrated for several mycophagous

oribatids and for T. putrescentiae (Smrz and Catska 2010).

For many years greenhouse crops were protected against soil fungi and arthropod pests

by soil-sterilizing chemicals, but this approach is now being phased out, because these

pesticides are highly toxic and pose a serious environmental risk. Solar sterilization, by

mulching with polyethylene sheets, developed to suppress pathogens, such as V. dahliae

and F. oxysporum f.sp. lycopersici (Katan et al. 1976) was also effective against bulb mites

at depths of 10, 20 and 30 cm underground (Gerson et al. 1981). While this approach is

more environmentally safe, it was less effective and like the first method, kills beneficial

soil organisms along with the pests. Another approach recently developed to control soil

pathogens is through suppressive soils using composts. By growing seedlings in plant

residue-based composts, Yogev et al. (2006) reduced disease development caused by

F. oxysporum in melon, tomato and cucumber, as compared with disease development in

the highly conducive peat, used in the present study. Adopting this methodology could

lower inoculum pressure of pathogenic fungi, thereby reducing the attraction of the plant

material to R. robini. Future studies, first in potted plant experiments and then in field trials,

are needed to evaluate this compost based suppressive soils approach for the control of

R. robini on germinating onion seedlings. However it must be emphasized that the stan-

dardization of compost preparation and maturity are crucial for the reproducibility of

results of these proposed experiments (Termorshuizen et al. 2006).

Acknowledgments We thank Dr. Ronny Cohen and Carmela Horev for their technical assistance, NabilOmeri for his collaboration and Prof. Uri Gerson and the two anonymous reviewers for their critical reviews.This paper is a contribution of the Agricultural Research Organization, Institute of Plant Protection, BetDagan, Israel.

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