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Biological Control

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The inhibitory role of dark septate endophytic fungus Phialocephala fortiniiagainst Fusarium disease on the Asparagus officinalis growth in organicsource conditionsSuronoa,b,c, Kazuhiko Narisawaa,b,⁎

aUnited Graduate School of Agriculture, Tokyo University of Agriculture and Technology, 3-8-1 Harumi, Fuchu, Tokyo 183-8538, Japanb College of Agriculture, Ibaraki University, 3-21-1 Chuo, Ami, Ibaraki 300-0393, Japanc Indonesian Agency for Agricultural Research and Development, Jl. Ragunan No.29, Pasar Minggu, Jakarta 12540, Indonesia

A R T I C L E I N F O

Keywords:Asparagus officinalisPhialocephala fortiniiFusarium oxysporum f.sp. asparagiCorn steep liquorOrganic soils

A B S T R A C T

Five of eight selected dark septate endophytic (DSE) fungal isolates had the ability to inhibit the growth ofFusarium oxysporum f. sp. asparagi in a dual culture test. We demonstrated that Asparagus officinalis inoculatedwith DSE fungi could survive and grow healthily without any typical disease symptoms after Fusarium diseasechallenge in inorganic or organic conditions in vitro. Phialocepahala fortinii isolate CKG.I.11 most effectivelypromoted the growth of A. officinalis after Fusarium disease challenge either with inorganic or organic sources.Inoculation of P. fortinii CKG.I.11 in A. officinalis plant on the organic nursery setting decreased the severity ofFusarium disease and increased the A. officinalis growth compared to A. officinalis challenged with F. oxysporumf.sp. asparagi and not inoculated with P. fortinii CKG.I.11 so that P. fortinii effectively promoted A. officinalisgrowth in Fusarium disease challenge.

1. Introduction

Asparagus officinalis is a long-term perennial vegetable crop withhigh market value and low input in its production (Nahiyan andMatsubara, 2012). Demand for this crop is increasing worldwide everyyear, but several factors limit its production, with one of the mainlimiting factors being Fusarium disease (Nahiyan and Matsubara, 2012;Waśkiewicz et al., 2013; Matsubara et al., 2014). Fusarium diseasesignificantly reduces A. officinalis production is mainly caused by Fu-sarium oxysporum f.sp. asparagi and Fusarium proliferatum (Reid et al.,2002; Nahiyan et al., 2011). Fusarium oxysporum f.sp. asparagi is thedominant pathogen of A. officinalis, especially in Japan (Nahiyan et al.,2011) causing root, stem, and crown rot (Elena, 2007, Nahiyan andMatsubara, 2012). This pathogenic fungus may persist during the as-paragus-free period for at least 25 years (Elena, 2007). Controlling thisdisease using chemicals has an impact on environmental pollution andthe disruption of agriculture ecosystems, but breeding for disease re-sistant plants requires high cost and a long time to develop (Pontaroliand Camadro, 2001). Therefore, an environmentally friendly and sus-tainable approach should be conducted for the control of this disease.One approach is to use dark septate endophytic (DSE) fungi as a bio-control agent for F. oxysporum f.sp. asparagi.

DSE fungi are a group of endophytic fungi that usually have mela-nized hyphae, form dark colonies in agar media, and make symbioticinteractions with plants (Jumpponen and Trappe, 1998, Thormannet al., 1999; Jumpponen, 2001). They colonize living plant root tissuesinter- and intracellularly but do not cause any typical disease symptomsin the plant (Jumpponen and Trappe, 1998; Knapp et al., 2012). Basedon the definition and category of symbiosis by De Bary (1879), thesymbiosis between DSE fungi and its host plants includes mutualism.Several studies have reported that DSE fungi have the potential as a bio-control agent for pathogenic fungi such as Heteroconium chaetospira thatsuppressed Verticillium yellows in Chinese cabbage (Narisawa et al.,2000) and Verticillium wilt in eggplant (Narisawa et al., 2002). Khastiniet al. (2012) reported that Veronaepsis simplex suppressed Fusariumdisease in Chinese cabbage. Other DSE fungal species such as Cadophorasp. effectively controlled Fusarium wilt on melon (Khastini et al., 2014),and Phialocephala sphareoides controlled Heterobasidion parviporum inNorway spruce (Terhonen et al., 2016). Tellenbach et al. (2013) de-monstrated that Phialocephala europaea significantly reduced thegrowth of Pytophthora citricola in vitro by producing secondary meta-bolites compounds. However, regarding the control of Fusarium diseasein A. officinalis caused by P. oxysporum f.sp. asparagi, the use of DSEfungi is still limited so that the focus of our research in this study was to

https://doi.org/10.1016/j.biocontrol.2018.02.017Received 25 November 2017; Received in revised form 16 February 2018; Accepted 21 February 2018

⁎ Corresponding author at: Department of Bioresource Science, College of Agriculture, Ibaraki University, 3-21-1 Chuo, Ami, Ibaraki 300-0393, Japan.E-mail address: kazuhiko.narisawa.kkm@vc.ibaraki.ac.jp (K. Narisawa).

select DSE fungus that suppresses Fusarium disease on A. officinalis.Therefore, further studies on the use of DSE fungi to control Fusariumdisease especially caused by F. oxysporum f.sp. asparagi are needed, toobtain the most effective DSE fungus to suppress this disease. Based onprevious research on the role of DSE fungi in suppressing plant diseases,we hypothesized that DSE fungi may have the potential to be used tosuppress Fusarium disease in A. officinalis.

The role of DSE fungi related to its effect in promoting A. officinalisgrowth in organic conditions and the preferences for organic source usesuch as organic N and P have been reported in our previous study(Surono and Narisawa, 2017). Therefore, further studies are needed toinvestigate whether selected DSE fungal isolates from our previousstudy are able or not able to promote the host plant in Fusarium diseasechallenge with various nutrient sources, especially organic source.There are only a few reports related to the association between DSEfungi and vegetable plant, such as asparagus, especially in plant diseasechallenge.

The aim of this study was to investigate the ability of DSE fungalisolates from our previous study (Surono and Narisawa, 2017) to sup-press Fusarium disease either in an inorganic or organic condition invitro systems and in an asparagus nursery setting. Testing in organicconditions, it is expected that the most effective DSE fungus may also beused as a biocontrol agent of Fusarium disease for A. officinalis organicfarming in the future. Organic asparagus cropping systems producebetter spear quality than conventional systems with agro-chemicalsources (Caruso et al., 2012). Asparagus officinalis as a host plant can beused as a valuable model to study microbial ecology and the biocontrolof pathogens because it is a low-input perennial crop and is affected byFusarium-related diseases (Yergeau et al., 2006). Therefore, the utili-zation of DSE fungi has the potential to overcome pathogen attack andpromote A. officinalis growth.

In organic A. officinalis cultivation, biocontrol agent to reduce theuse of pesticides and decomposer to produce organic fertilizer to re-place the use of inorganic fertilizers are needed (Espejo et al., 1997;Kibblewhite et al., 2008; Monokrousos et al., 2008; Xu et al., 2014). Thesupply of organic fertilizer can be obtained from composting of A. of-ficinalis biomasses such as twigs and leaves when thinning in winter andcan be used other sources such as manure when the initial planting toharvest time is over a long period of cultivation (Monokrousos et al.,2008; Xu et al., 2014). The DSE fungal isolates of our previous study tobe used in the present study are known to be potential as decomposersof soil organic matter (Surono and Narisawa, 2017) and if the presentstudy is able to suppress Fusarium disease in A. officinalis, the DSEfungal isolates will play a significant role in promoting growth andproduction of A. officinalis organically in the future because they have adouble ability as a growth promoter, decomposer of soil organic matterand Fusarium disease controller in A. officinalis.

2. Material and methods

2.1. Dark septate endophytic and pathogenic fungal material

Eight DSE fungal isolates were used that promote A. officinalisgrowth in previous studies, comprising P. fortinii isolates III.Pi.I8,CKG.I.1.1, CKG.I.10.3, CKG.I.11, CKG.II.10.1, CYA.II.14.3, CYA.II.17,and unidentified isolate sp. CKG.IV.10 (Surono and Narisawa, 2017).All DSE fungal isolates were obtained from Prof. Kazuhiko Narisawa.Fusarium oxysporum f.sp. asparagi NBRC 31382 used as a pathogen of A.officinalis in this study was obtained from NBRC (National Institute ofTechnology and Evaluation – Biological Resource Center, Japan). AllDSE fungal and pathogen fungal isolates were grown on potato dextroseagar (PDA) media.

2.2. Antagonism between Fusarium oxysporum f.sp. asparagi and darkseptate endophytic fungal isolates

An agar plug (5mm diameter) of DSE fungal isolates was placed onPDA 6 cm distance away from F. oxysporum f.sp. asparagi. Due to theslow growth of DSE fungi, the fungi were firstly grown for 2 weeks,subsequently the fungal pathogen was grown in the same Petri dish asthe DSE fungal isolates for dual culture testing. Fusarium oxysporum f.sp.asparagi was grown alone in a Petri dish as a control. The measurementfinished when one of the control plates was fully covered with colony(Terhonen et al., 2016). After 2 weeks, the inhibition zone and inhibi-tion of the radial growth of the pathogen were measured (Maciá-Vicente et al., 2008). The experiment was repeated three times. Theradial colony growth of F. oxysporum f.sp. asparagi towards the antag-onistic fungus (Ri) and that on a control (Rc) dish were measured andthe colony growth inhibition was calculated using the formula:(Rc− Ri)/Rc× 100 (Lahlali and Hijri, 2010).

2.3. Effect of dark septate endophytic fungal isolates to promote Asparagusofficinalis growth in Fusarium disease challenge in the inorganic and organicsource conditions

Experiments were conducted to determine the effects of DSE fungiinoculation on A. officinalis growth with Fusarium disease challenge ininorganic and organic conditions in vitro. The compositions of themedia used for this test are shown in Table 1. The procedure for thesetreatments was in accordance with previous procedures in Surono andNarisawa (2017). Corn steep liquor (CSL; Nature Aid; Sakata Seed,Yokohama, Japan) used in the organic media treatment contained 3%nitrogen (N), 3% phosphorus (P) and 2% potassium (K). A. officinalisseeds were surface sterilized by immersion in 70% ethanol for 1.5 minand then a solution of sodium hypochlorite (1% available chlorine) for3min. The seeds were rinsed three times with sterilized distilled water,then dried overnight, and ready to be used in next experiment (Suronoand Narisawa, 2017). Then, A. officinalis seedlings with or without DSEfungal inoculation were challenged with F. oxysporum f.sp. asparagigrown on water agar. A. officinalis seedlings were overlaid onto thefungal colony and incubated for 3 weeks in the same conditions(Khastini et al., 2012). There were three replicates for each treatment,including the control.

2.4. Effect of Phialocephala fortinii isolate CKG.I.11 to promote A.officinalis growth and suppress Fusarium disease in an organic nurserysetting

This experiment was conducted to investigate the ability of P. fortiniiisolate CKG.I.11 as the best promoter of A. officinalis growth in previoustreatments to promote A. officinalis growth and suppress Fusarium dis-ease in an organic nursery setting. The treatments consisted of 1) A.officinalis was not inoculated with P. fortinii CKG.I.11 and was not in-fected with F. oxysporum f.sp. asparagi as a control, 2) A. officinalis was

Table 1Composition of media used for inoculation of Asparagus officinalis with dark septate en-dophytic fungal isolates with Fusarium oxysporum f.sp. asparagi challenge on the inorganicand organic conditions.

Constituent Inorganic Organic

KH2PO4 1.5 0NaNO3 1.0 g 0MgSO4·7H2O 1.0 g 0Corn steep liquor (CSL)* 0 1mLOatmeal 10 g 10 gAgar 15 g 15 gDistilled water 1000mL 1000mL

* CSL contained 3% nitrogen (N), 3% phosphorus (P) and 2% potassium (K).

Surono, K. Narisawa

infected with F. oxysporum f.sp. asparagi, 3) A. officinalis was inoculatedwith P. forinii isolate CKG.I.11 and was infected with F. oxysporum f.sp.asparagi, and 4) A. officinalis was inoculated with P. fortinii isolateCKG.I.11 and was not infected with F. oxysporum f.sp. asparagi. Eachtreatment consisted of ten A. officinalis seedlings and three replications.

The experiment was conducted using organic soil from the FieldScience Center (FSC), Ibaraki University, Japan. The soil properties oforganic soil sample from FSC were pH 6.37 (slightly acidic soil), 4.31%C, 0.41% N and C/N 10.6. The organic soil samples were air-dried,sieved through a 5mm mesh, autoclaved at 121 °C for 30min, and re-autoclaved after 24 h using with same procedure. The P. fortiniiCKG.I.11 inoculum was prepared as described by Usuki et al. (2002).Subsequently, 5% of P. fortinii CKG.I.11 inoculum was added and mixedwith the sterilized soil samples in each treatment. Surface-sterilized A.officinalis seed was transplanted into pots contain 25 g soil. An in-oculum of F. oxysporum f.sp. asparagi was prepared in accordance withKhastini et al. (2014). Seedlings without fungal inoculum were as thecontrols. The procedure for surface sterilization of A. officinalis seed wassame as previously (Section 2.3). All seedling treatments were in-cubated for 4 weeks at room temperature with an 18 h: 6 h (L: D) re-gimen and intensity of 180molm−2 s−1 and watered with distilledwater every 3 days. After incubation for 1week, the nursery seedlingsoil was supplemented with 0.1% corn steep liquor as a sole organicnutrient. Then, after incubation time for 2 weeks, treatments 2 and 4were infected with F. oxysporum f.sp. asparagi, then disease symptomswere observed every week using the disease index of Narisawa et al.(2000) until 8 weeks. The A. officinalis plants were harvested after8 weeks of infection with F. oxysporum f.sp. asparagi and oven-dried at35 °C for five days, and the roots were used for recording DSE fungalcolonization. The dry weight of the biomass of each treatment with P.fortinii CKG.11 or F. oxysporum f.sp. asparagi was measured and wascompared with that of the uninoculated controls (Surono and Narisawa,2017).

2.5. Anatomical observations, assessment of root colonization, and re-isolation of DSE fungal isolate from Asparagus officinalis root tissues

To determine the effects and the colonization degree of selected DSEfungi on the roots of A. officinalis to inhibit Fusarium disease, 5-week-old asparagus seedling roots and 56-day-old asparagus seedlings rootswith DSE fungal inoculation and Fusarium disease challenge in vitro andin a nursery with setting using organic soil media were washed, cross-sectioned, and stained with 50% acetic acid solution containing 0.005%cotton blue, before being examined using a light microscope (BX51;Olympus, Tokyo, Japan). The calculation of the percentage of DSEfungal colonization referred to the method described by Usuki et al.(2002). Stained A. officinalis roots were examined along grid lines andeach grid cell was designated as either colonized or non-colonized

(Narisawa et al., 2004).The DSE fungal re-isolation was determined according to methods

described by Narisawa et al. (2002). Inoculated DSE fungi were re-isolated from A. officinalis roots by washing with Tween 20 three timesand sterile water three times, next the sterilized roots were driedovernight. Root segments were placed on 50% corn meal agar. Forty-five root segments were chosen randomly from each treatment. Thefrequency of re-isolation was calculated as the mean number of rootsegments colonized by the fungus (Narisawa et al., 2002).

2.6. Data analysis

The mean dry biomass of each treatment was calculated and ana-lyzed using one-way ANOVA with R version 3.0.2 (The R Foundationfor Statistical Computing Platform, Vienna, Austria). Differences amongtreatment means were detected using Tukey’s Honestly SignificantDifference test (Tukey HSD).

3. Results

3.1. Antagonism between Fusarium oxysporum f.sp. asparagi and darkseptate endophytic fungal isolates

Five of eight DSE fungal isolates inhibited the growth of F. oxy-sporum f.sp. asparagi (Fig. 1). The inhibition levels by P. fortinii isolatesIII.Pi.I8, CKG.I.1.1, CKG.I.11, CKG.II.10.1, and unidentified isolate sp.CKG.IV.10 on F. oxysporum f.sp. asparagi were 75.1%, 74.9%, 67.8%,65.7%, and 79.0% (Table 2), respectively. The other three DSE fungalisolates (P. fortinii isolates CKG.I.10.3, CYA.II.14.3, and CYA.II.17) didnot inhibit the growth of F. oxysporum f.sp. asparagi.

Fig. 1. Phialocephala fortinii CKG.I.11 and unidentified isolate CKG.IV.10 inhibited the growth of Fusarium oxysporum f.sp. asparagi in a dual culture test. Arrows indicate inhibition zoneof DSE fungi with F. oxysporum f.sp. asparagi colony growth.

Table 2Growth inhibition of Fusarium oxysporum f.sp. asparagi by dark septate endophytic fungalisolates in an antagonism assay using a dual culture method.

DSE fungal isolate Origin Percentage inhibition ofpathogen growth rate (%)

Phialocephala fortiniiIII.Pi.I8

Rubus sp. root 75.1bc*

P. fortinii CKG.I.1.1 Chamaecyparis obtusaroot

74.9bc

P. fortinii CKG.I.11 C. obtusa root 67.8bcP. fortinii II.10.1 C. obtusa root 65.7bUnidentified isolate

CKG.IV.10C. obtusa root 79.0c

P. fortinii CKG.I.10.3 C. obtusa root 0aP. fortinii CYA.II.14.3 C. obtusa root 0aP. fortinii CYA.II.17 C. obtusa root 0a

* Values within columns followed by the same letter are not significantly different(P < 0.05) after Tukey’s Honestly Significant Difference test.

Surono, K. Narisawa

3.2. Inoculation of Asparagus officinalis with dark septate endophyticfungal isolates and Fusarium oxysporum f.sp. asparagi under inorganic andorganic conditions in vitro

In the agar media with inorganic conditions, all DSE fungal isolatespromoted A. officinalis growth with Fusarium disease challenge (Fig. 2).Asparagus officinalis grew without any disease symptoms and normallycompared with control plants in which the leaves began yellowing(Fig. 3). All DSE fungal isolates suppressed Fusarium disease up to 100%in organic conditions (Table 3). The growth rates versus the control of P.fortinii isolates CKG.I.11, CKG.II.10.1, CKG.I.1.1, III.Pi.I8, CYA.II.14.3,CYA.II.17, CKG.I.10.3, and unidentified isolate CKG.IV.10 were 240%,198%, 189%, 179%, 171%, 142%, 141%, and 127%, respectively. As-paragus officinalis inoculated with P. fortinii isolate CKG.I.11 had thehighest dry weight compared with other DSE fungal treatments.

In the treatment using 0.1% CSL as an organic nutrient source, the

control plants did not grow well and were stunted and showed appearany disease symptoms, whereas A. officinalis inoculated with P. fortiniiisolates CKG.I.11, CYA.II.17. CKG.I.1.1 and CKG.I.10.1 could growwithout any disease symptoms (Fig. 5) and suppressed Fusarium diseaseup to 100% in organic conditions (Table 3). Whereas, the diseasesuppression of A. officinalis inoculated with P. fortinii isolatesCKG.I.10.3, CYA.II.14.3, III.Pi.I8, and unidentified isolate CKG.IV.10were 93.3%, 72.2%, 70%, and 78.9%, respectively (Table 3). Comparedwith control plants, the growth rates of A. officinalis inoculated with P.fortinii isolates CKG.I.11, CYA.II.17. CKG.I.1.1, CKG.II.10.1, CKG.I.10.3,CYA.II.14.3, III.Pi.I8, and unidentified isolate CKG.IV.10 were 474%,449%, 407%, 380%, 286%, 175%, 169%, and 204%, respectively(Fig. 4). In this treatment, P. fortinii isolate CKG.I.11 also gave thehighest A. officinalis dry weight compared with other organic treat-ments (Fig. 4).

Fig. 2. The effect of dark septate endophytic fungi onAsparagus officinalis growth with Fusarium disease challengein inorganic agar media incubated at 23 °C with a 16-h: 8-h(L: D) photoperiod (180molm−2 s−1) for 3 weeks. Valuesare means ± SD, n=3. Means with the same letter werenot significantly different (P < 0.05) after Tukey’sHonestly Significant Difference test.

Fig. 3. Performances of Asparagus officinalis inoculated withDSE fungal isolates with Fusarium oxysporum f.sp. asparagichallenge on inorganic agar media. A) Control plant (witharrow to show yellowing effect on asparagus leaves), B)treatment with Phialocephala fortinii isolate CKG.I.11, C)treatment with P. fortinii isolate CKG.II.10.1, and D) treat-ment with P. fortinii isolate CKG.I.1.1 inoculation incubatedat 23 °C with a 16-h: 8-h (L: D) photoperiod(180mol m−2 s−1) for 3 weeks.

Surono, K. Narisawa

3.3. Inoculation of Phialocephala fortinii to promote Asparagus officinalisand suppress Fusarium disease in a nursery setting using organic soil

Fusarium oxysporum f.sp. asparagi caused an increase in disease se-verity in A. officinalis. The length of incubation time with the highestseverity at 56 days after inoculation of F. oxysporum f.sp. asparagireached 44% (Fig. 6). Whereas A. officinalis treated with P. fortiniiisolate CKG.I.11, the disease severity was lower even decreased duringincubation from 42 to 56 days after inoculation with F. oxysporum f.sp.asparagi reached 1.33% (Fig. 6). The performance of A. officinalistreated with P. fortinii isolate CKG.I.11 appeared healthy and grew wellcompared with A. officinalis treated with F. oxysporum f.sp. asparagiwithout treatment with P. fortinii isolate CKG.I.11 (Fig. 7).

Inoculation of A. officinalis with P. fortinii caused enhancement thegrowth in all treatments either with or without Fusarium challenge, andeven P. fortinii was effective in Fusarium-challenged asparagus (Fig. 8).Phialocephala fortinii-inoculated asparagus increased dry weight sig-nificantly eventhough it was challenged with Fusarium disease com-pared with P. fortinii-untreated asparagus (Fig. 8). As well as treatmentswithout Fusarium challenge, dry weight of. A. officinalis increased in theP. fortinii inoculation treatments compared with uninoculated treat-ments (Fig. 8).

The colonization rate of P. fortinii in A. officinalis roots with Fusariumdisease challenge in a nursery setting using organic soil reached 46.7%,whereas in the treatment with only inoculation with P. fortinii isolateCKG.I.11 without Fusarium disease challenge was higher and reached53.3%. Fusarium oxysporum f.sp. asparagi colonization at the root ofcontrol plants reaches 100%, roots grew abnormally and tend to adwarf. While F. oxysporum f.sp. asparagi colonization of A. officinalisinoculated with P. fortinii and infected with F. oxysporum f.sp. asparagi

only reached 12.5%. Phialocephala fortinii isolate CKG.I.11 penetratedthe cortical tissues of A. officinalis roots and formed microsclerotia-likeforms (Fig. 9). The results of re-isolation of P. fortinii in A. officinalisseedling with and without Fusarium disease challenge in a nurserysetting were 22.2% and 26.7%, respectively.

3.4. Anatomic observation of fungal colonization

Heavy colonization of P. fortinii isolate CKG.I.11 occurred in A. of-ficinalis roots especially in epidermal cells in inorganic and organicsource conditions in vitro, and it may protect A. officinalis roots fromFusarium disease (Fig. 9). However, the colonization pattern did notreach the vesicular systems of A. officinalis roots. Phialocephala fortiniiisolate CKG.I.11 formed darker microsclerotia forms inside corticaltissues of A. officinalis roots in the treatment with Fusarium diseasechallenge than in treatment without Fusarium disease challenge in anursery setting using organic soil (Fig. 9).

4. Discussion

We demonstrated that five of eight selected DSE fungal isolates hadthe ability to inhibit the growth of F. oxysporum f.sp. asparagi in dualculture tests (Table 2; Fig. 1). Slow-growing endophytic fungi includingDSE fungi are known as antifungal producers and have the potential tobe fungal pathogen inhibitors (Sieber, 2002; Mandyam andJumpponen, 2005; Maciá-Vicente et al., 2008; Terhonen et al., 2016).However, investigations on the ability of DSE fungi to inhibit fungalpathogen growth and to produce antifungal compounds are still limited.Antagonism between F. oxysporum f.sp. asparagi and DSE fungal isolatesin this study may be because of biologically active compounds

Table 3The inhibition role of Phialocephala fortinii isolate CKG.I.11 on the disease severity and suppression of Fusarium disease in Asparagus officinalis in inorganic and organic conditions in the invitro system.

DSE fungal isolates Disease severity (%) Disease suppression (%)

Inorganic Organic Inorganic Organic

Phialocephala fortinii III.Pi.I8+ Fusarium oxysporum f.sp. asparagi 0a* 30d* 100a* 70d*

P. fortinii CKG.I.1.1+ F. oxysporum f.sp. asparagi 0a 0a 100a 100aP. fortinii CKG.I.11+ F. oxysporum f.sp. asparagi 0a 0a 100a 100aP. fortinii II.10.1+ F. oxysporum f.sp. asparagi 0a 0a 100a 100aUnidentified isolate CKG.IV.10+ F. oxysporum f.sp. asparagi 0a 21.1c 100a 78.9cP. fortinii CKG.I.10.3+ F. oxysporum f.sp. asparagi 0a 6.67b 100a 93.3bP. fortinii CYA.II.14.3+ F. oxysporum f.sp. asparagi 0a 27.8d 100a 72.2dP. fortinii CYA.II.17+ F. oxysporum f.sp. asparagi 0a 0a 100a 100aF. oxysporum f.sp. asparagi 53.3b 100e – –

* Values within columns followed by the same letter are not significantly different (P < 0.05) after Tukey’s Honestly Significant Difference test.

Fig. 4. The effect of dark septate endophytic fungi toAsparagus officinalis growth with Fusarium disease challengeon organic agar media incubated at 23 °C with a 16-h: 8-h(L: D) photoperiod (180molm−2 s−1) for 3 weeks. Valuesare means ± SD, n=3. Means with the same letter werenot significantly different (P < 0.05) after Tukey’sHonestly Significant Difference test.

Surono, K. Narisawa

produced in media by these DSE fungi (Castillo et al., 2002) and had adetrimental effect on pathogen growth. Although they are the samespecies, P. fortinii isolates CKG.I.10.3, CYA.II.14.3, and CYA.II.17 didnot inhibit the growth of F. oxysporum f.sp. asparagi in dual culturetests. These P. fortinii isolates may have the potential to induce A. of-ficinalis to come resistant to Fusarium disease attack by other mechan-isms when associated with plants. In the next treatment, P. fortinii alsopromoted A. officinalis growth in inorganic and organic source condi-tions (Fig. 2, Fig. 4). As in the previous study by Khastini et al. (2012),although there was no zone of inhibition at the point contact betweenDSE fungus Veronaeopsis simplex and F. oxysporum in dual culture tests,the DSE fungus suppressed Fusarium disease in Chinese cabbage. Incontrast, Terhonen et al. (2016) reported that although the endophyticfungus Cryptosporiopsis sp. was extremely effective in inhibiting Het-erobasidion parviporum growth in dual culture tests, but this endophytehad detrimental effects on Norway spruce seedlings. However, in this

study, we reported that P. fortinii isolates III.Pi.I8, CKG.I.1.1, CKG.I.11,CKG.II.10.1, and unidentified isolate CKG.IV.10 have the double abilityto inhibit F. oxysporum f.sp. asparagi growth in agar medium and topromote A. officinalis growth during pathogen attack (Table 2, Table 3,Fig. 1, Fig. 2, Fig. 4). Therefore, these DSE fungal isolates have thepotential to be used as a biocontrol agent for F. oxysporum f.sp. asparagi.

We demonstrated that A. officinalis inoculated with DSE fungi couldsurvive and grow healthily without any typical disease symptoms withFusarium disease in inorganic or organic nutrient conditions in vitro andpromoted A. officinalis growth compared with the control plants. Theeffect of each treatment using DSE fungi was significant different fromthe control, but all DSE fungi were able to promote and protect A. of-ficinalis growth in disease conditions. The other mechanism of Fusariumdisease protection cannot be explained in detail from this study, but aprevious study with dual culture tests showed that several DSE fungalisolates have the ability to suppress the growth of F. oxysporum f.sp.

Fig. 5. Performances of Asparagus officinalis inoculated withDSE fungal isolates with Fusarium oxysporum f.sp. asparagichallenge on organic agar media. A) Control plant (witharrow to show yellowing effect on asparagus leaves), B)treatment with Phialocephala fortinii isolate CKG.I.11, C)treatment with P. fortinii isolate CYA.II.17, and D) treatmentwith P. fortinii isolate CKG.I.1.1 inoculation incubated at23 °C with a 6-h: 8-h (L: D) photoperiod (180molm−2 s−1)for 3 weeks.

Fig. 6. The inhibition role of Phialocephala fortinii isolateCKG.I.11 on the disease severity index of Fusarium disease inAsparagus officinalis in organic nursery setting. Mean valuesthat differ significantly at each time point (P < 0.01) areindicated by asterisk after Tukey’s Honestly SignificantDifference test, n=10. Foa= F. oxysporum f.sp. asparagi,Pf= Phialocephala fortinii. Bars indicate standard deviationof means.

Surono, K. Narisawa

asparagi in agar media (Table 2). The compact colonization of DSEfungal hypha also occurred in A. officinalis roots in these treatments(Fig. 9). These abilities may make a contribution to Fusarium diseasereduction in A. officinalis inoculated with DSE fungi. Other DSE fungalisolates such as P. fortinii isolate CKG.I.10.3 and CYA.II.17 that had noability to reduce F. oxysporum f.sp. asparagi growth, but it could pro-mote the growth of A. officinalis either in inorganic or organic sourcemedia, so there are may be other mechanisms whereby this fungus canimprove the resistance of A. officinalis to Fusarium disease. In contrast,Schulz and Boyle (2005) stated that endophytic fungi may cause diseasesymptoms when inoculated with a non-original host plant, but ourprevious study (Surono and Narisawa, 2017) demonstrated that theselected DSE fungi did not produce any typical disease symptoms in A.officinalis as a non-original host plant. Furthermore, in the current studywe demonstrated these DSE fungi also promoted A. officinalis growth indisease conditions. Reported previously, the selected DSE fungal iso-lates used in this study were originally from Chamaecyparis obtusa(Japanese cypress). He et al. (2002) reported that induced systemic

resistance developed when A. officinalis inoculated with non-pathogenicfungi was challenged with F. oxysporum and caused a reduction in rootlesions from 50% to 25%. Arbuscular micorrhizal fungi species Glomusintraradices increased Fusarium root rot tolerance in A. officinalis byindirectly induced resistance of A. officinalis roots to that disease beforethe pathogen colonization (Matsubara et al., 2003). In the previousstudy by Narisawa et al. (2002), P. fortinii suppressed Verticilium dahliaeand protected eggplant from the attack by this fungal pathogen withcolonization of eggplant root tissues. Jumpponen (2003) stated thereare three mechanisms related to the role of DSE fungi in inhibitingfungal pathogens or minimizing the negative effect of pathogens inplant growth and performance. i.e., competition for plant photo-synthates or for colonization sites, production of pathogen inhibitorycompounds, and colonization of host plants by DSE fungi developedinducing plant defense responses to plant infection. We reported for thefirst time that P. fortinii isolate CKG.I.11 was the best isolate amongother DSE fungal isolates to promote A. officinalis growth either withinorganic or organic nutrient sources in Fusarium disease in vitro. When

Fig. 7. The inhibition role of Phialocephala fortinii isolateCKG.I.11 on Fusarium disease in A. officinalis seedlings usingorganic soil. A) A. officinalis seedlings were challenged withF. oxysporum f.sp. asparagi in organic conditions. The seed-lings became yellowed and severely damaged. B) A. offici-nalis seedlings were treated with P. fortinii isolate CKG.I.11and challenged with F. oxysporum f.sp. asparagi did not showany typical external symptoms, grew well and appeared tobe healthy.

Fig. 8. The effect of Phialocephala fortinii CKG.I.11 onAsparagus officinalis growth with Fusarium disease challengein a nursery setting using organic soil media and 0.1% CSL,as an organic source, incubated at 23 °C with a 16-h: 8-h (L:D) photoperiod (180molm−2 s−1) for 3months in a growthchamber. Values are means ± SD, n=10. Means with thesame letter were not significantly different (P < 0.05) afterTukey’s Honestly Significant Difference test.

Surono, K. Narisawa

this isolate was applied to promote A. officinalis in a nursery settingwith a disease challenge using organic soil media, and which con-sistently showed positive effects on A. officinalis growth with or withoutthe pathogen infection. The disease severity of A. officinalis treated withP. fortinii isolate CKG.I.11 decreased in the period of 45–56 days ofpathogen incubation time and A. officinalis that had shown symptoms ofthe disease grew normally again by producing new healthy shoots. Itwas indicated that P. fortinii isolate CKG.I.11 potentially protected A.officinalis from pathogen attack and promoted plant growth especiallyin an organic nursery setting.

Heavy colonization by DSE fungi occurred in A. officinalis roots in-oculated with DSE fungal isolates and reached the cortical cells of roots.In the organic nursery setting, hyphae of P. fortinii isolate CKG.I.11penetrated the cortical cells and formed microsclerotia inside the A.officinalis root both with or without pathogen attack, but in the pre-sence of pathogen, the microsclerotia-like forms of P. fortinii isolateCKG.I.11 appeared darker (Fig. 9). Peterson et al. (2008) stated thatcolonization by some DSE did not invade the vascular cylinder or causedegradation of cortical and vascular parenchymal cell of plants. Heavycolonization indicated a protection pattern from DSE fungi for A. offi-cinalis, so that it became tolerant to Fusarium disease. The heavy

colonization in roots allowed the aboveground parts of A. officinalis togrow normally with higher dry weight either in inorganic or organicmedia compared with the control plant. There were no typical diseasesymptoms appearing on A. officinalis. In contrast to the effect of theinteractions between fungal pathogens and plants, the interaction ofmutualistic fungal endophytes by root colonization provides manybeneficial effects for the plant, such as systemic protection against pa-thogen attack (Liu et al., 2007). Colonization by endophytic fungi fillsout an ecological niche and left no space for fungal pathogens, and theprocess including penetration of epidermal and cortical cells withoutcausing any disease symptoms in the plants, even it in the event ofpathogen attack (Dutta et al., 2014). Endophytic fungi are generallyconsidered to protect plants through rapid colonization and thus ex-haust the limited available substrates, leaving nothing available forpathogens to grow (Pal and Gardener 2006). Colonization by en-dophytic fungi usually offers protection to plants in various ways, suchas the production of pathogen inhibitor compounds, ecological nicheoccupations used by pathogens, or interference with cellular membranepathogens by producing chitinase, cause cell death in pathogens(Ganley et al., 2008; Shittu et al., 2009; Waqas et al., 2014). This studyshowed that P. fortinii isolate CKG.I.11, both in in vitro treatment and in

Fig. 9. Image of A. officinalis roots challenged withFusarium oxysporum f.sp. asparagi and treated withPhialocepahala fortinii CKG.I.11 on inorganic and organicagar media in the in vitro system (A-C) and in the nurserysetting (D-E). The image in (A) shows uninoculated rootsunder Fusarium disease challenge with Fusarium oxy-sporum f.sp. asparagi hyphae (arrows) as a control treat-ment, (B) A. officinalis roots colonized by P. fortiniiCKG.I.11 hyphae (arrows) under Fusarium disease chal-lenge in inorganic conditions, (C) A. officinalis roots co-lonized by P. fortinii CKG.I.11 hyphae (arrows) under or-ganic conditions. DSE fungal colonization can be seen onthe root surface and within epidermal cells (Ep) and thecortex (Co). Note the absence of DSE fungal colonizationin the vascular cylinder (Vc). Bars= 20 μm. Images of A.officinalis seedling roots colonized by P. fortinii CKG.I.11with (D) and without (E) Fusarium disease challenge inorganic nursery setting. Phialocephala fortinii CKG.I.11hypha formed in the cortical tissues (arrows) and formedmicrosclerotia-like form (asterisk). Phialocephala fortiniiisolate CKG.I.11 formed darker microsclerotia forms in-side cortical tissues of A. officinalis roots in the treatmentwith Fusarium disease challenge than in treatment withoutFusarium disease challenge. Bars= 10 μm.

Surono, K. Narisawa

a nursery setting, actively colonized the roots of A. officinalis that havean impact on healthy asparagus growth and to protect it from pathogenattack.

In conclusion, P. fortinii isolate CKG.I.11 most effectively promotedthe growth of A. officinalis with Fusarium disease either in inorganic ororganic sources conditions in vitro and in a nursery setting using organicsoil media. In the nursery setting, P. fortinii isolate CKG.I.11 suppressedFusarium disease severity. Therefore, P. fortinii isolate CKG.I.11 has thepotential for further testing in the field to determine the consistency ofits ability to promote the growth of A. officinalis and to suppressFusarium disease attack on A. officinalis organically.

Acknowledgements

This work was partly supported by Japan Society for The Promotionof Science (JSPS) KAKENHI Grant Number 17H03948 (to KN). Thefunding source had no role in study design; in the collection, analysisand interpretation of data; in the writing of the report; and in the de-cision to submit the article for publication.

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