Journal of Soil and Plant Biology

ISSN: 2652-2012

Research Article

Fungistatic Effect of a Chicken Manure-Based Organic Fertilizer for Suppression of a Soilborne Pathogen Rhizoctonia Solani Kühn

Xiaowei Pan1, Jeanne D Mihail1, Robert J Kremer2 and Xi Xiong1*

1Division of Plant Sciences, University of Missouri, Columbia, MO, USA

2School of Natural Resources and Division of Plant Sciences, University of Missouri, Columbia, MO, USA

Received: 17 May 2019

Accepted: 07 June 2019

Version of Record Online: 13 June 2019

Citation

Pan X, Mihail JD, Kremer RJ, Xiong X (2019) Fungistatic Effect of a Chicken Manure-Based Organic Fertilizer for Suppression of a Soilborne Pathogen Rhizoctonia Solani Kühn. J Soil Plant Biol 2019(1): 61-72.

Correspondence should be addressed to
Xi Xiong, USA

E-mail: xiongx@missouri.edu
DOI: 10.33513/JSPB/1901-07

Copyright

Copyright © 2019 Xi Xiong et al. This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and work is properly cited.

Abstract

Repeated fungicide applications on intensively managed turf can result in the development of fungicide-resistant fungal strains, cause a financial burden for producers, and raise concerns for environmental safety. Alternatively, organic fertilizers might have a secondary benefit that increases soil microbial populations and hence reduces soilborne disease. However, it is unclear if organic fertilizers might contain indigenous microorganisms that directly suppress soilborne pathogens, or if soil microbial communities will need to be altered in a long-term effort and thus suppress fungal diseases. Therefore, the objective of this study was to evaluate the effect of a chicken manure-based organic fertilizer for control of Rhizoctonia solani Kühn that causes Zoysiagrass (Zoysia japonica Steud.) large patch, and to determine its possible mechanism. An in vitro experiment found that addition of the chicken manure-based organic fertilizer directly inhibited R. solani mycelial growth on potato dextrose agar up to 10 days after treatment, suggesting a potential fungistatic effect likely due to the beneficial bacteria the fertilizer contains. Two 4-week growth chamber experiments further confirmed that application of the organic fertilizer consistently restricted fungal colonization on fresh Zoysiagrass shoots by up to 75%, compared to the control. This effect was comparable to a conventional fungicide treatment, and was possibly partially attributed to the increased bacteria group in the soils which received the organic fertilizer treatment. Collectively, our results indicate a possible fungistatic effect from the chicken manure-based organic fertilizer and its potential inclusion in a disease control program to minimize the use of conventional fungicides.

Keywords 

Fungicide Resistance; Large Patch; Organic Turf Management; Turfgrass Disease Management; Zoysiagrass

Introduction

Zoysiagrass (Zoysia japonica Steud.) is a perennial turfgrass widely used in golf courses, parks, and residential lawns in the United States and Asian countries [1]. As a warm-season (C4) species, it exhibits supreme tolerance to extreme temperatures and adaptation to low maintenance [2,3]. Compared to other turf species, Zoysiagrass has relatively high tolerance to most fungal diseases, except large, patch caused by Rhizoctonia solani Kühn AG 2-2 LP [4]. Disease symptoms appear to be large-size patches with straw-color grasses that may vary from less than 0.3 meter to more than 6 meters in diameter. Environmental conditions that favor the development of large patch include thatch layer temperatures between 15 and 25°C, excessive and prolonged duration of surface wetness, and compacted and poorly drained soils [4]. In the transition zone of the United States, large patch occurrence is most common in early spring and mid- to late-fall, when the weather is cool and wet.

Fungicide applications throughout the entire growing season are often necessary for acceptable disease control on intensively managed turf. There are, however, concerns regarding such a practice. Excessive or repeated fungicide applications can risk environmental and human health [5,6] and increase the selection of fungicide resistance [7], a major problem for more than 40 years [8]. Brent and Hollomon [9] stated that fungicide applications led to resistance from survival and spread of initial and rare mutants, and avoiding repetitive and sole use of fungicide is one of the major strategies to manage fungicide resistance. Additionally, some fungicides can directly affect soil health by reducing beneficial soil microbial populations, such as mycorrhizal fungi [10,11]. Therefore, there is a need to develop alternative approaches for minimizing the potential impact of fungicide applications on the environment while maintaining acceptable disease control.

Soil organic amendments, such as compost, manure, and plant residue and waste, have been used as organic fertilizers on turf with a potentially added benefit of reducing turfgrass diseases. In principle, organic fertilizers may boost soil microbes that suppress pathogens by competing for colonization and/or infection sites, nutrients, or by producing pesticidal compounds [12,13]. Liu et al., [14] found that incorporation of organic fertilizers including hydrolyzed poultry feather meal, blood meal, wheat (Triticum aestivum L.) germ, potassium sulfate and bone meals resulted in increases of soil bacterial and fungal populations up to 2.9 and 2.8 times, respectively. The authors also found up to 71% reduction of dollar spot (caused by Sclerotinia homoeocarpa F. T. Bennett) incidence on organic fertilizer treated creeping bentgrass (Agrostis stolonifera L.), compared to the non-treated control. Similarly, Nelson [15] reported that application of composted turkey (Meleagris gallopavo Linnaeus) litter resulted in 12% less Pythium root rot (caused by Pythium graminicola Subraman.) on creeping bentgrass, along with a 9-fold increase in the soil bacterial population compared with the nontreated control. Collectively, these reports suggest a possible relationship between an increase in soil microbial population and a decrease in disease incidence.

For conventional fertilizers, however, previous research suggested a negative correlation between soil microbial population and conventional fertilizer usages. Treseder [16] summarized results from 82 published studies, and found that nitrogen addition from conventional sources caused an average of 15% reduction on microbial biomass, with a greater decline of the microbial abundance followed a greater addition of nitrogen in mass and/or application duration.

There is an inconsistency in the literature, however, regarding the linkage between utilizing organic fertilizer and disease suppression through alteration of soil microbial populations. For example, Ryu et al., [17] found that applications of composted livestock manure showed no effect on large patch suppression in four of the five locations examined. At the only location where significant large patch suppression was observed, the authors could not attribute such an effect to altered soil microbial populations, as evidenced by no changes found in culturable soil microbial group counts. In contrast, Pan et al., [18] found that repeated application of a chicken (Gallus gallus domesticus Linnaeus) manure-based organic fertilizer over two growing seasons suppressed large patch on a Zoysiagrass fairway by 49%, compared with the untreated control. A shift in soil bacterial group to a Gram-negative bacteria dominance was also observed following the organic fertilizer application. By comparison, repeated application of a sewage based-organic fertilizer resulted in no such effects on either disease suppression or soil microbial community alternation after two growing seasons. In this experiment, the authors also discovered that the chicken manure-based fertilizer contained 1.8-fold more bacteria group than the sewage based-fertilizer, including 1.7-fold greater Pseudomonas fluorescens [18], a known Gram-negative bacterium with antifungal properties [19,20]. Moreover, Aryantha et al., [21] stated that chicken manure is the only manure among cow (Bos Taurus L.), sheep (Ovis aries L.), and horse (Equus ferus caballus L.) manures that stimulated endospore forming bacteria, which is strongly related to the white lupin (Lupinus albus) seedling survival from root rot, dieback, and plant death resulted from Phytophthora cinnamomi infection.

Altering soil microbial populations to agronomically significant levels is a time-consuming process which often requires efforts in multiple seasons under field conditions following repeated treatment applications [22]. Therefore, it is uncertain whether the disease suppression observed by Pan et al., [18] following application of the chicken manure-based organic fertilizer was mainly due to the beneficial microbes indigenous to the fertilizer, or to the stimulation of certain soil microbial groups over-time, or both. Hence, the objective of this research was to determine the possible mechanism by which chicken manure based-organic fertilizer interacts with the soilborne pathogen that causes large patch on Zoysiagrass turf in the short-term.

Materials and Methods

In vitro study

An isolate of R. solani AG 2-2 LP, originating from large patch infected Zoysiagrass in Columbia, Missouri, was used in the in vitro study. The fungal isolate was confirmed as multinucleate R. solani by morphological traits described in Parmeter and Whitney [23] and Paulitz and Schroeder [24], and by staining fungal nuclei with 4'6-Diamidino-2-Phenylindole (DAPI) as described in Cooke et al. [25]. Confirmation of R. solani to the anastomosis group of AG 2-2 LP was performed by PCR using AG 2-2 LP specific primers designed by Toda et al. [26].

The isolate was maintained on Potato Dextrose Agar (PDA; Difco BD, Erembodegem-Aalst, Belgium) medium in petri plates at 4°C, subcultured to fresh PDA plates and incubated at 24°C in the dark for 4 days prior to treatment application. The PDA medium was prepared by adding 39 g PDA to 1 liter distilled deionized water with additions of 100 ppm ampicillin and 100 ppm streptomycin sulfate [27].

A 6-mm-diameter agar plug was removed from the margin of the R. solani AG 2-2 LP colony, and transferred to the center of an 8.5 cm diameter PDA plates. Immediately after transfer, a chicken manure-based organic fertilizer (CM; Chicken Manure, Back to Nature Inc., Slaton, TX), a synthetic N fertilizer (SN; UMAXX, Koch Agronomic Services, LLC, Wichita, KS), or a synthetic fungicide (SF; active ingredient azoxystrobin, Heritage, Syngenta Crop Protection Inc., Greensboro, NC) was applied with 3 ml autoclaved distilled deionized water to the agar surface, in addition to a Nontreated Control (NC). Based on the manufacture’s information, the CM is an odorless granular product developed from fully composted chicken manure under an aerobic condition with temperatures approaching 71°C for over four months.

The rates for CM and SN were 1,500 and 160 kg ha-1, respectively, to provide the same amount of N equivalent to 75 kg ha-1 according to their labels of 5% and 47% N (w/w), respectively. The amount of N in CM was also confirmed at the University of Missouri Soil Testing Lab prior to treatment applications. In addition to N, the CM used in this experiment also contained minimal amounts of P (0.8%), K (5.9%) and Mg (6.2%), in addition to trace amounts of Zn and Cu. All treatments, including the NC, were applied to 6 replicate plates. After application, plates were then incubated at 24°C in the dark, and the colony diameter of R. solani was measured daily from the center to the edge of the colony on three different axes to generate an average for two weeks.

For the in vitro study, treatments were arranged in a completely randomized design with six replications, and the entire experiment was repeated once. Levene’s test was used to confirm the homogeneous variances between the two repeated experiments. Data from the two experiments were then combined for ANOVA by PROC GLM procedure in SAS 9.4 (SAS Institute, Cary, NC).

Growth chamber study

The inocula of R. solani AG 2-2 LP were prepared from a mixture of six isolates that were isolated from large patch-infected Zoysiagrass from local golf course fairways in Columbia, Missouri, following the procedure described by Green et al. [4]. All isolates were confirmed to be R. solani AG 2-2 LP using the same procedures described above. Preparation of the inocula was performed using organic wheat (Triticum aestivum L.) grains following the procedure described by Carling and Sumner [28], and stored at 4ºC for future use. Briefly, 500 g wheat grains were soaked in distilled deionized water overnight before autoclaving at 121ºC for 30 min and cooled for 6 hours. The autoclaved wheat grains were then transferred to sterilized flasks for future use. The six R. solani isolates were subcultured to PDA plates for 5 days, then 6-mm-diameter agar plugs were removed from PDA plates by a sterilized tool and transferred to the flasks with autoclaved wheat grains. A sterilized long stirring tool was used to stir the wheat grains to ensure a good mixture with the agar plugs that contain the fugal culture. The openings of the flasks were then sealed by cotton balls and aluminum foil. The wheat grains were stirred every 3 days and kept at 24ºC in dark for 2 weeks before use.

Zoysiagrass ‘Meyer’ plugs were removed by shovel to the depth of 18 cm from the South Farm Research Center at Columbia, Missouri (38.91ºN, 92.28ºW), then shaped to fit into pots with 15 cm diameter and 18 cm depth. The soil was a Mexico silt loam (fine, smectitic, mesic Vertic Epiaqualfs), with 2% organic matter and pH at 7.1. The area was free of large patch, and received no fungicide application in the current growing season. The plants were allowed to grow in greenhouse conditions with irrigation as needed to prevent drought. A 24N-8P-16K fertilizer was applied weekly to provide 12 kg N ha-1 for 4 weeks, before pots were transferred to a growth chamber at 25/20ºC for day and night temperatures, with a 14 h photoperiod at a Photosynthetically Active Radiation (PAR) of 500 μmol·m–2·s–1. The pots were re-arranged within the chamber every other day to minimize the location effect in the growth chamber. The Zoysiagrass was cut once per week to maintain 5 cm height, and fertilized with 24N-8P-16K to provide 12 kg N ha-1 weekly. Distilled deionized water was applied every other day to saturate the rootzone soil until water drained out from the bottom of the pots.

Immediately after transfer to the growth chamber, three randomly selected basal leaf sheath samples and four soil samples from 0-2, 2-4, 4-6, and 6-10 cm depth were collected from each pot and placed on water agar plates for one week. No presence of R. solani was detected for either leaf sheath or soil samples according to the morphological traits of R. solani [23,24]. One week after transfer to the growth chamber, 25 g R. solani inocula were spread evenly on the soil surface beneath the plant canopy of each pot. In addition, 25 g autoclaved wheat grains without R. solani colonization were added to the non-inoculated control. As disease developed and symptoms reached about 15% of canopy (approximately 12 days after inoculation), curative application of predesigned treatments were applied evenly onto the soil surface. Treatments applied included CM, a chicken manure-based organic fertilizer, SN, a synthetic N fertilizer, and SF, a synthetic fungicide, as described above for the in vitro experiment. Additionally, treatments also included two controls, with one being no treatment added to the inoculated pots (NC), and a non-inoculated control with addition of autoclaved wheat grains (CA) as described above. All treatments were applied as one single application to four replicate pots, which resulted in a total of 20 pots.

The rates of CM (1,500 kg ha-1) and SN (160 kg ha-1) were determined to provide the equivalent N according to the N contents described on their labels. The SF was applied at 0.6 kg ha-1 active ingredient based on the label suggestions, by using a CO2-pressurized backpack sprayer equipped with TeeJet 8004 (Spraying Systems Inc., Wheaton, IL) flat fan nozzles calibrated to deliver 561 L ha-1 at 275 kPa. Immediately following treatment application, pots were irrigated with distilled deionized water until the water drained from the bottom of the pots, except fungicide treated pots which were watered 24 h after application.

Weekly evaluations included large patch incidence (%) that was assessed visually, and clipping biomass after oven drying at 85ºC for 3 days. From each pot, two soil cores with 2 cm diameter and 5 cm depth were collected using a soil probe at 0, 1, 2, and 4 Weeks After Treatment (WAT). The soil cores were first broken apart and passed through a 2 mm sieve, before mixing them thoroughly and freezing at -20ºC. Soil microbial population was determined by soil Phospholipid Fatty Acids (PLFA) according the procedure described by Buyer and Sasser [29]. Briefly, 2 to 2.5 g soil samples were added to Teflon-lined screw cap culture tubes and freeze-dried before extraction of and esterification of the fatty acids. The purified PLFA extracts were dissolved in hexane and identified on a gas chromatograph (Model 6890, Agilent Technologies) with an autosampler, split-splitless inlet, and flame ionization detector coupled to an Agilent Ultra 2 column. The peak responses were translated into molar responses using an internal standard, and were compared against a database of known microbial PLFA fingerprints (Sherlock Microbial Identification System; MIDI, Inc.). The Agilent Chemstation software (Agilent Technologies) was used to identify and quantify individual PLFA peaks, and to assign individual PLFA biomarkers to microbial groups and fatty acid types [18].

Microbial groups detected included total fungi (Sum of 16:0, 18:0, 18:1ω9, and 18:2ω6; [30]), total bacteria (Sum of i14:0, i15:0, a15:0, 15:0, i16:0; 10Me16:0, i17:0, a17:0, cy17:0, 17:0, br18:0, 10Me 17:0, 18:1ω7, 10Me18:0, and cy19:0; [31]), gram-positive bacteria (Sum of i14:0, i15:0, a15:0, 15:0, i16:0, i17:0, and cis18:1ω9; [32]), gram-negative bacteria (Sum of 16:1ω9, cy17:0, 18:1ω5, 18:1ω7, and cy 19:0; [33]), actinobacteria (Sum of 10Me16:0, and 10Me18:0; [34]), and Arbuscular Mycorrhizal (AM) fungi (16:1ω5; [35]). General characterization of community composition was based on ratios calculated for Fungi to Bacteria (F/B) and Gram-positive to Gram-negative bacteria (G+/G–). The ratios of cyclopropyl 17 to its monoenoic precursor (Cy17/pre; 17:0cy/16:1ω7c; [36]), and saturated to monounsaturated fatty acids (Sat/mono; Sum of 12:0, 13:0,14:0,15:0, 16:0, 17:0,18:0, and 20:0 / sum of cis16:1ω11, cis16:1ω9, cis16:1ω7, cis16:1ω5, cis17:1ω9, cis17:1ω8, cis17:1ω7, and cis17:1ω5; [36]) were calculated as shifts in these ratios are known to occur when some bacteria experience environmental stresses (i.e., low C, pH changes, low moisture, high temperature) [36]. Total PLFA concentration (nmole g−1 soil) was determined as the total microbial biomass [37], and the abundance of microbial groups was reported as mole percent (mol %) of total PLFA concentration.

A second growth chamber experiment was carried out to monitor the development of large patch by sampling twenty 1-cm basal leaf sheaths from randomly selected shoots that did not show disease symptoms in each pot at 0, 1, 2, and 4 WAT, and determining the presence of R. solani on water agar plates. This was performed to avoid possible influence of destructive sampling from the first growth chamber experiment. Hence, a separate set of Zoysiagrass plants were prepared and processed in the same manner as described above, and grown simultaneously under the same conditions as described above. Treatment combinations, including the five treatments described above and four sampling timings (i.e., 0, 1, 2 and 4 WAT), were applied to four replicate pots as described, which resulted in a total of 80 pots. After random sampling, the basal leaf sheath samples were placed on water agar plates, and the plates were monitored up to one week for determining the presence of R. solani by fungal morphological traits [23,24].

In both growth chamber experiments, all treatments were arranged in a completely randomized design with four replications. The data were analyzed by PROC GLM procedure in SAS 9.4 (SAS Institute, Cary, NC). Significant means were separated using Fisher’s Protected LSD at P≤0.05.

Results and Discussion

In vitro study

Mycelia of R. solani grew fairly quickly in the NC plates, and within 4 days they covered the entire plate (Figure 1). In plates containing SN, the fungal mycelia showed a slightly slower initial growth compared to the NC, but reached the same growth rate by 4 Days After Treatment (DAT). As expected, the SF was the only treatment that completely inhibited R. solani AG 2-2 LP mycelial growth during the two-week period. The plates containing CM, in comparison, showed a 100% inhibition of mycelia growth for 2 days (Figure 2), and then followed by a significant reduction of fungal colony diameter in the range of 19-72% compared to NC plates between 2 to 10 DAT (Figure 1). This result suggests that CM might have a direct fungistatic effect against R. solani.

Fungistatic-Effect-of-a-Chicken-Manure-Based-Organic-Fertilizer-for-Suppression-of-a-Soilborne-Pathogen-Rhizoctonia-Solani-Kühn

Figure 1: Mycelial growth of Rhizoctonia solani AG 2-2 LP on Potato Dextrose Agar (PDA) influenced by surface application of a Chicken Manure-based organic fertilizer (CM; Chicken Manure), a Synthetic N fertilizer (SN; UMAXX), a fungicide (SF, azoxystrobin), in addition to a Non-treated Control (NC). Vertical bars above the days after treatments represent the Fisher’s Protected LSD at P=0.05.

Fungistatic-Effect-of-a-Chicken-Manure-Based-Organic-Fertilizer-for-Suppression-of-a-Soilborne-Pathogen-Rhizoctonia-Solani-Kühn

Figure 2: Mycelial growth of Rhizoctonia solani AG 2-2 LP at three days after treatment influenced by surface application of a chicken manure-based organic fertilizer (CM; chicken manure), a Synthetic N fertilizer (SN; UMAXX), a fungicide (SF, azoxystrobin), in addition to a Non-treated Control (NC). The blue circles indicate the edge of the mycelial growth marked every day. There was only one circle marked on plates received CM as no growth was observed in the first two days.

Similar to other organic fertilizers, manure-based fertilizer typically contains a substantial amount of microorganisms compared to synthetic materials [38]. The CM used in this experiment contained a total microbial biomass of 281 nmole g−1, determined by PLFA. Among them, 45% were grouped as bacteria, including 8.1% of actinomycetes and 5.4% of Pseudomonas fluorescens, and 39% were identified as fungi [18]. In addition to competing for colonization sites and nutrients with fungi [13], the gram-negative bacterium P. fluorescens can effectively suppress soilborne diseases by producing antifungal metabolites such as HCN, siderophores, protease, 2,4-diacetylphloroglucinol, fluorescent pigments including pyrrolnitrin, or by stimulating the activity of chitinase and peroxidase [19,20]. Some gram-positive bacteria and actinobacteria, are also known as biological control agents which suppress soilborne disease [39]. Collectively, these results indicate that the chicken manure-based fertilizer utilized in this experiment has a direct suppressive effect again R. solani, and this fungistatic effect is mostly likely attributed to the beneficial microorganisms containing in this organic fertilizer.

It is important, however, to recognize that manure-based fertilizers might contain antibiotic residues [40], and their usage in agricultural production might introduce or induce bacteria that are resistant to antibiotics [41]. Although compared to swine (Sus spp.) and beef cattle manure, poultry litter generally contains lower concentrations of antibiotics for the major antibiotics used in production [40], ecological risk of animal waste varies substantially based on specific antibiotics and their dosages used. For example, chicken manure collected at 7 days after amoxicillin administration at 100 mg/kg is considered safe to the environment, while ciprofloxacin at 100 mg/kg requires 10 days withdrawal period before its excretion in manure drops to the minimum [42]. Those antibiotics, however, can be effectively removed through the process of composting. Composting involves aerobic digestion which is commonly employed in the agricultural industry. A recent review summarized related studies and found that composting after 42-45 days can remove majority, ranging between 93.8% and 98.5%, of the tested antibiotics from chicken manure [40]. The CM included in the current experiment was a product after 4-month composting, strongly indicating its low risk to the environment.

Large patch incidence

The first growth chamber experiment revealed that disease incidence, assessed as percent large patch, varied among treatments. Over time, large patch started to spread out in the NC treatment (Figures 3 and 4), with the most rapid increase of 2.2 times from 0 to 1 WAT (Table 1). As expected, pots which received the SF treatment maintained a steady, low disease pressure during the 4-week period, and resulted in only 41% of large patch coverage compared to the incidence in NC at 4 WAT. The treatment with CM showed slower disease development especially in the first two weeks (Figure 3), and resulted in 27% and 21% reductions in large patch coverage compared to NC at 1 and 2 WAT, respectively. This result supports the findings from the in vitro experiment where addition of CM suppressed the R. solani growth for up to 10 days (Figure 1). The treatment with SN also showed a similar effect in reducing large patch development, supporting an early report by Rodrigues et al., [43] who found that the incidence of Rhizoctonia root rot on beans (Phaseolus vulgaris L.) caused by R. solani AG-4 was reduced by 18% following ammonium sulfate treatment. Since the in vitro study found no fungicidal effect from SN when plants were absent (Figure 1), it is speculated that the disease suppression observed in this experiment was due to the stimulated growth of the Zoysiagrass plants, which subsequently masked the disease symptoms.

Fungistatic-Effect-of-a-Chicken-Manure-Based-Organic-Fertilizer-for-Suppression-of-a-Soilborne-Pathogen-Rhizoctonia-Solani-Kühn

Figure 3: Representative Zoysiagrass plants at 2 weeks after treatment in growth chamber influenced by surface application of a Chicken Manure-based organic fertilizer (CM; chicken manure) and a Non-treated Control (NC).

Fungistatic-Effect-of-a-Chicken-Manure-Based-Organic-Fertilizer-for-Suppression-of-a-Soilborne-Pathogen-Rhizoctonia-Solani-Kühn

Figure 4: A close exam of Zoysiagrass shoot infected by Rhizoctonia solani AG 2-2 LP, showing a black-colored lesion at the basal leaf sheath and dieback from the leave tips (left), and mycelia on the surface of an infected leaf (right) observed under a microscope.

 

 

Large patch incidence (%)

Treatmentz

0 WAT

1 WAT

2 WAT

3 WAT

4 WAT

CM

15aAy

24bB

30cB

38dC

41dC

SN

16aA

24bB

28bcB

30cB

25bcB

SF

18aA

16aA

18aA

18aA

19aA

NC

15aA

33bC

38bcC

40cC

46dC

Table 1: Zoysiagrass large patch incidence (%) influenced by treatments when assessed 0, 1, 2, 3, and 4 Weeks After Treatment (WAT). The treatments applied to inoculated plants included a Chicken Manure-based organic fertilizer (CM), a Synthetic N fertilizer (SN), a Synthetic Fungicide (SF), and a Non-treated Control (NC).

zResults from non-inoculated control with autoclaved wheat grains (CA) were not included in this table because there was no disease development for this treatment during this experiment.

yMeans followed by the same lowercase letter within the same row were not significantly different based on the Fisher’s Protected LSD (P=0.05); Means followed by the same capital letter within the same column were not significantly different based on Fisher’s Protected LSD (P=0.05).

Clipping biomass

Without the presence of disease, pots which received CA treatment maintained an overall steady shoot growth from 0 to 4 WAT (Table 2). Compared to pots which received only inoculation (NC), CA-treated pots yielded a total of 0.5 g more clipping biomass during the 4-week period, supporting a previous report that large patch reduces shoot growth likely due to the vascular blockage caused by crown lesions [44]. Treatment with SF did not affect clipping biomass from 0 to 4 WAT, reflecting the constant disease pressure found in table 1. Application of SN resulted in an increase of shoot growth over time, and showed 37% and 43% greater clipping biomass production compared to NC at 3 and 4 WAT, respectively (Table 2). The increased clipping biomass at a later stage may have resulted from the urease inhibitor in SN which slows down the release of N from the urea. Compared to NC, CM-treated pots produced 29% greater clipping biomass initially, but this effect quickly diminished and no differences were found in clipping biomass during the rest of the experiment. It is worth noting, however, that CM-treated plants maintained the same or greater shoot growth rate for up to 3 WAT compared to plants receiving SF treatment, despite the presence of disease symptoms.

 

Clipping biomass (g)

Treatment

0 WAT

1 WAT

2 WAT

3 WAT

4 WAT

CM

0.62bCz

0.60bBC

0.46aA

0.52abA

0.44aA

SN

0.44aA

0.57bcABC

0.54abAB

0.70dB

0.67cdC

SF

0.46aAB

0.48aA

0.48aAB

0.54aA

0.56aBC

NC

0.48aAB

0.51aAB

0.48aAB

0.51aA

0.47aAB

CA

0.56abBC

0.66bC

0.59abB

0.55aA

0.60abC

Table 2: Zoysiagrass clipping biomass (g) assessed at 0, 1, 2, 3, and 4 Weeks After Treatment (WAT). The treatments applied on inoculated plants included a Chicken Manure-based organic fertilizer (CM), a Synthetic N fertilizer (SN), a Synthetic Fungicide (SF), and a Non-treated Control (NC), in addition to a non-inoculated control with autoclaved wheat grains (CA).

zMeans followed by the same lowercase letter within the same row were not significantly different based on the Fisher’s Protected LSD (P=0.05); Means followed by the same capital letter within the same column were not significantly different based on Fisher’s Protected LSD (P=0.05).

Colonization of leaf sheaths by R. solani

The number of basal leaf sheath samples colonized by R. solani, collected from the second growth chamber experiment, was influenced by treatments applied. In the absence of inoculation, treatment CA resulted in no colonization as expected during this period (Table 3). With the presence of disease, pots receiving NC treatment showed a steady increase of shoots colonized by R. solani in the first two weeks, and resulted in 77% greater amount of infected shoots at 2 WAT compared to 0 WAT. Treatment with SF resulted in no R. solani colonization at 1 and 2 WAT, and 85% reduction of R. solani colonization compared to NC at 4 WAT (Table 3). This result supports a typical 2 to 4-week re-application interval suggested by the fungicide label. Plants treated with SN showed no changes in the amount of freshly infected shoots during the entire experiment, similar to that of the NC treatment at all sampling times. This result again confirmed that addition of SN has no direct effect restricting R. solani colonization. The reduced disease incidence observed in table 1 likely resulted from an N effect stimulating shoot growth (Table 3) and hence, “masking” disease symptoms. Compared to NC, however, treatment with CM consistently showed reduced R. solani colonization, and resulted in 56, 65, and 75% less infected new shoots compared to NC at 1, 2, and 4 WAT, respectively (Table 3). These results corroborate with findings in the in vitro experiment (Figure 1), and collectively suggest a fungistatic effect of CM against R. solani.

 

The number of R. solani colonized basal leaf sheathz

Treatment

0 WAT

1 WAT

2 WAT

4 WAT

CM

1.3aBy

0.8aA

0.8aA

0.5aA

SN

1.5aB

2.0aB

2.0aB

2.0aB

SF

1.5bB

0.0aA

0.0aA

0.3aA

NC

1.3aB

1.8abB

2.3bB

2.0abB

CA

0.0aA

0.0aA

0.0aA

0.0aA

 Table 3. The number of Zoysiagrass basal leaf sheaths colonized by Rhizoctonia solani AG 2-2 LP at 0, 1, 2, and 4 Weeks After Treatment (WAT). The treatments applied on inoculated plants included a Chicken Manure-based organic fertilizer (CM), a Synthetic N fertilizer (SN), a Synthetic Fungicide (SF), and a Non-treated Control (NC), in addition to a non-inoculated control with autoclaved wheat grains (CA).

zTwenty leaf sheath samples that did not show disease symptoms were randomly collected from each of the four replicate pots for each treatment at each sampling time.

yMeans followed by the same lowercase letter within the same row were not significantly different based on the Fisher’s Protected LSD (P=0.05); Means followed by the same capital letter within the same column were not significantly different based on Fisher’s Protected LSD (P=0.05).

Soil microbial community and composition

No treatment effect was noticed for the total microbial biomass as detected by PLFA, although the PLFA results varied in different weeks. At 0 WAT, the total PLFA was determined to be 277 nmol g-1, and it increased by 63% and 77% at 1 and 2 WAT, respectively. By 4 WAT, however, the total PLFA dropped to 364 nmol g-1, resulting in a 26% decrease compared to the microbial biomass determined at 4 WAT. This result indicates that the microbial growth might have been limited over a longer period of time likely due to the limited resources in the individual pots.

Most of the microbial groups and stress indicators assessed varied in time, but they were not significantly affected by treatments applied or interactions between treatment and sampling time (data not shown). However, treatments applied influenced the bacteria, actinobacteria, AM fungi, and the stress indicator Cy17/pre in the soil microbial community (Table 4). Introduction of R. solani inocula in the NC pots reduced the total bacteria by 2% during this 4-week period, compared to pots that received no inoculation (CA). This effect, however, was reversed when N fertilizer was applied, regardless of the source of N, i.e., synthetic or organic sources. Application of fungicide, however, did not affect the total amount of the bacteria in the soil compared to NC. Bittman et al., [45] reported that dairy manure application in spring providing 200 kg N ha-1 increased up to 55% of soil bacteria per year compared to control on a grassland. This increase in soil bacteria was associated with up to 40% reduction in soil fungi every year during a three-year period, indicating the competitive or antagonistic relationship between the soil bacteria and fungi. In the present experiment, only application of CM elevated soil bacteria significantly compared to the NC treatment, further indicating its likelihood of fungistatic effect against soilborne fungi.

Treatment

Bacteria

Actinobacteria

AM fungi

Cy17/pre

 

−−−−−−−−−−−−−−−−−mol %−−−−−−−−−−−−−−−−−

CM

50.5bcz

8.3a

4.8a

0.49b

SN

49.9abc

8.3a

4.9a

0.46ab

SF

49.8ab

8.1a

5.1a

0.47ab

NC

49.5a

8.2a

5.0a

0.41a

CA

50.7c

9.0b

5.4b

0.42a

Table 4: Treatment main effect on soil bacteria, actinobacteria, Arbuscular Mycorrhizal (AM) fungi, and cyclopropyl 17/monoenoic precursor ratio (Cy17/pre; mol%). The treatments applied on inoculated plants included a Chicken Manure-based organic fertilizer (CM), a Synthetic N fertilizer (SN), a Synthetic Fungicide (SF), and a Non-treated Control (NC), in addition to a non-inoculated control with autoclaved wheat grains (CA).

zMeans followed by the same letter within the same column were not significantly different based on Fisher’s Protected LSD (P=0.05).

The actinobacteria proportion was not different among treatments after the inoculation during the 4-week period (Table 4), suggesting that none of the treatments significantly influenced this microbial group. Compared to the non-inoculated soil in CA, inoculated soils showed a decrease in actinobacteria up to 10%, indicating a possible competition or antagonism between R. solani and the actinobacteria.

Similar to the actinobacteria response, the AM fungi proportion was not affected by the treatments with inoculation (Table 4). Introduction of R. solani reduced the AM fungi group up to 11%, compared to the non-inoculated soil in CA. The AM fungi are considered beneficial to plants, aiding nutrient uptake by the roots, especially phosphorus, from soil [46-48]. Additionally, previous research reported that the addition of AM fungi suppressed diseases caused by R. solani [49]. Collectively, our results again pointed out a possible competition between the AM fungi and the R. solani inoculant.

Treatments did not affect the Cy17/pre ratio compared to the non-inoculated control, except the CM which increased the ratio by 20% (Table 4). It has been suggested that increased Cy17/pre ratio serves as an indicator of a relatively more stressful soil environment for bacteria, likely due to substrate/nutrient deficiency [50]. In the present experiment, the elevated bacterial group following CM application might have resulted in greater competition for substrates and/or nutrients in a confined system (i.e., individual pots).

When all fungal species were combined, the total fungal group was significantly influenced by the interaction of treatment and sampling time (Table 5). After inoculation but before treatment application (0 WAT), soils which received R. solani inocula exhibited the same or greater amount of total fungi compared to the soil which received no inoculation. When R. solani was absent, soil in the CA treatment maintained a constant fungal group during the 4-week period. In all inoculated soils, however, a general decline trend was found overtime, and consequently, no differences in total fungi were found among inoculated and non-inoculated soils between 1 and 4 WAT. This result might reflect the competition in a confined system between R. solani that was introduced, and the indigenous soil microorganisms over this 4-week period. Although no statistical differences were detected when comparing fungal group at each sampling time, treatment with SF and CM resulted in a rapid reduction of total fungi starting in 1 or 2 WAT, respectively. In contrast, the declining fungi in SN or NC treatments did not occur until 4 WAT. This result corroborated with findings presented in tables 1-3 and from the in vitro experiment, which collectively indicated a possible fungistatic effect of CM.

 

Fungi (mol %)

Treatment

0 WAT

1 WAT

2 WAT

4 WAT

CM

24.7cBCz

24.2bcA

22.2abA

21.7aA

SN

23.1bAB

24.3bA

23.0bA

19.6aA

SF

25.9cC

23.0bA

22.5abA

20.5aA

NC

23.0bAB

23.5bA

22.8bA

20.6aA

CA

21.2aA

22.4aA

22.4aA

21.1aA

Table 5: Total fungal response (mol %) in the soil microbial community assessed at 0, 1, 2, and 4 Weeks After Treatment (WAT). The treatments applied included a Chicken Manure-based organic fertilizer (CM), a Synthetic N fertilizer (SN), a Synthetic Fungicide (SF), and a Non-treated Control (NC), in addition to a non-inoculated control with autoclaved wheat grains (CA).

zMeans followed by the same lowercase letter within the same row were not significantly different based on the Fisher’s Protected LSD (P=0.05); Means followed by the same capital letter within the same column were not significantly different based on Fisher’s Protected LSD (P=0.05).

Conclusion

The results of the present studies suggest that fungicide application provides the best large patch suppression as expected, but application of N fertilizer, regardless of organic or synthetic sources, also reduces large patch severity compared to the control. While the conventional N fertilizer suppresses large patch by boosting shoot growth and subsequently masking disease symptoms, the chicken manure-based organic N fertilizer directly inhibits R. solani mycelial growth when in contact, likely due to the beneficial bacteria it contains. Using this organic N fertilizer also increased the bacterial proportion in the soil microbial community, and consequently minimized the colonization of R. solani onto healthy Zoysiagrass plants. Collectively, our results suggest a potential fungistatic effect of the chicken manure-based organic fertilizer and benefits for incorporating organic fertilizer as well as synthetic N fertilizer to manage large patch. Subsequent field studies are needed to assess the efficacy of these integrated approaches for developing a control program in Zoysiagrass turf in order to reduce our dependency on conventional fungicides.

Acknowledgements

We would also like to thank the Daniel F. Millikan Endowment for providing the graduate fellowship.

Compliance with Ethical Standards

Funding: This material is based on work that was supported by the National Institute of Food and Agriculture, USDA, under award no. 1006256.

Conflict of Interest: Author X. Pan declares that she has no conflict of interest. Author J.D. Mihail declares that she has no conflict of interest. Author R.J. Kremer declares that he has no conflict of interest. Author X. Xiong declares that she has no conflict of interest.

Ethical Approval: This article does not contain any studies with animals performed by any of the authors. This article does not contain any studies with human participants performed by any of the authors.

Informed Consent: Informed consent was obtained from all individual participants included in the study.

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