Inoculation of Corn Seedlings with Piriformospora indica Influences Grain Biomass Yield, Forage Quality, Rumen Degradation Kinetics and Fourier Transformed Infrared Spectroscopy Molecular Structures

Document Type : Research Articles

Authors

1 Department of Animal Science, Malayer University, Malayer, Iran

2 Department of Agronomy and Plant Breeding, Malayer University, Malayer, Iran

3 Department of Animal Science, College of Agriculture, Isfahan University of Technology, Isfahan, Iran

Abstract

Piriformospora indica (P. indica) as a root growth promoting endophyte may improve corn grain production and forage quality in ruminant under semi-arid climate condition. The aim of current study was to evaluate the influence of corn seedling- P. indica endophyte inoculation on grain and forage biomass yield, nutritive value and Fourier transformed infrared spectroscopy (FTIR) of molecular structures. Corn seedlings were inoculated with P. indica and were grown under natural condition and were compared with control group (12 experimental boxes per each treatment). Half of the experimental boxes from each treatment selected to make corn silage and the other half remained for grain biomass yield. Inoculation with P. indica increased grain biomass yield (P=0.05) but had no impact on chemical composition except for total carbohydrate content which tended to be greater for P. indica-inoculated group (p <0.10). Leaf to stem ratio decreased (P=0.04) and ether extract increased in silages from treated corn compared with control group (P=0.04). In situ ruminal dry matter and organic matter degradability at 24 h of ruminal incubation were greater in P. indica corn grain from inoculated samples compared with control grain samples (P=0.02). Silage from P. indica group had greater in vitro rate of gas production (p <0.05) and tended to have greater volume of gas produced until 24 h of incubation (P=0.06). The FTIR ratio between amid I to amid II and amid II to lignin area tended to decrease in silages from P. indica group compared with control group samples (P=0.09). The principle component analysis differentiated the FTIR area related to carbohydrate between experimental groups. In conclusion, under semi-arid climate condition inoculation of corn seedlings with P .indica may improve the feeding value of corn seed and silage in ruminants.

Keywords


INTRODUCTION

Corn grain and corn silage are widely used in ruminants feeding due to their high carbohydrate content, good palatability and great energy value (NRC, 2001). Low digestibility of forage is mainly reflected in low voluntary intake and low energy supply that could limit milk and meat production of dairy cattle (Jung and Allen, 1995). However in the arid and semi-arid areas, it is a challenge to produce high quality grain and forage crops due to the abiotic stresses which restrict growing potential of crops (Waller et al. 2005). Increased plant productivity and crop improvement might therefore, rely on high chemical input and which is made at the expense of a huge negative impact on the environment (Chapin Iii et al. 2000; Parmesan and Yohe, 2003). An alternative to the use of chemicals is the application of genetically-modified crops, although these also exhibit their own drawbacks and controversies (Mannion and Morse, 2013). For this reason, abiotic-stress tolerance can be evoked in crops by the exploitation of worldwide abundant endophytic arbuscular mycorrhiza fungi, which live in reciprocally beneficial relationships with 80% of land plants (Newman and Reddell, 1987). Piriformospora indica (P. indica), a root endophyte fungus belonging to the Sebacinales in the Basidiomycota shows the increase in nutrient uptake and utilization in plant. The P. indica has a unique property and can be easily cultured axenically on a variety of medium (Varma et al. 1999). P. indica has a wide host range, including members of the Brassicaceae, which has been found to be involved in promoting the growth of various plants, including cereal crops such as rice, wheat, barley and corn, as well as many Dicotyledoneae (Verma et al. 1998; Peškan-Berghöfer et al. 2004; Ghabooli et al. 2013). In the study of Kumar et al. (2009), Zhang et al. (2018), Wu et al. (2018) and Hosseini et al.(2018) corn plant colonized with P. indica increased the corn resistant to biotic and abiotic stresses and simultaneously also the biomass grain yield. None of those studies investigated the effect of corn seed inoculation with P. indica on its feeding value in ruminants. To our knowledge, only one research has been done on the influence of P. indica on the quantitative and qualitative characteristics of plants for use in the livestock feeding. This research showed that hay of alfalfa seedlings inoculated and co-inoculated with root growth promoting microorganisms had improved nutritional value compared with hay from non-treated alfalfa seedlings, and co-inoculation was the most effective, however, changes were relatively minor (Jafari et al. 2018). Molecular structures of feed cannot be determined using traditional wet chemical analysis, while Fourier transformed infrared spectroscopy (FTIR) can be used to reveal these structures including primary protein molecular structures (e.g. amide I, amide II), secondary protein structures (e.g. α-helix and β-sheet), lipid and carbohydrate molecular structures (Yu, 2005). The hypothesis of current study was that P. indica-inoculated corn plant may have improved corn grain and corn silage feeding value in ruminant. Therefore, the aims of this study were to use chemical composition analysis, in situ ruminal degradation, in vitro gas production and FTIR to evaluate the effect of corn seedlings inoculation with P. indica on corn grain and silage nutritive value in ruminants.

 

MATERIALS AND METHODS

Culture of root endophyte fungus, P. indica

P. indica was cultured and maintained on CM (complex medium) (Pham et al. 2008) at 24 ˚C for 4 weeks. This medium contains mineral salts, microelements, and also complex components such as peptone, yeast extract and casein hydrolysate (Pham et al. 2008). Former researches revealed that CM is one of the best mediums for P. indica culture. For solid medium, 15 g l-1 agar was used. The spore suspension was prepared (Ghabooli et al. 2013).

 

Co-culture of P. indica with corn seedling

For plant inoculation, corn seeds of the cultivar “640” were surface-sterilized with 70% ethanol (v/v) for 30 s followed by 6% sodium hypochlorite (NaCl) for 10-15 min. Then, they were rinsed in water, and were germinated for 2-3 days at 18/04/2014. Corn seedlings were inoculated by immersing in the spore suspension solution (adjusted to 5×105 spores per mL) with gentle shaking for 1 h. The other group of seedlings (control) was dipped in sterile water only. Inoculated and non-inoculated corn seedlings were later transferred into boxes under natural weather condition at Malayer University experimental farm (34.3020˚ N, 48.8146˚ E), filled with normal soil at date of 04/05/2014, two weeks after inoculation. The experiment was arranged in a completely randomized design with two fungus treatments (inoculated and non-inoculated) in twelve biological replications (n=12 per each treatment).

 

Microscopic analysis of colonization

Root samples from P. indica-inoculated and control plants were collected 14 days after plant inoculation. Root were softened by 10% KOH solution for 15 min, acidified with 1 M HCl for 10 min, and stained with 0.02% Trypan blue overnight, and then were distained with 50% lacto-phenol for 1 h prior to microscopic observation under a light microscope (Ghabooli et al. 2013).

 

Corn silage preparation

Among twelve experimental boxes, six boxes selected randomly for silage making and six boxes reminded to harvest corn as grain. Whole plants from six boxes from each of treatment were harvested when kernel milk was around 50% at date of 26/08/2014, and weighed to measure the total biomass yield. Harvested plants chopped to 1-cm length with a portable chopper (Nasr Company, Isfahan). Adequate samples used to measure dry matter (DM) content of fresh forages at 50 ˚C for 48 h. The rest of wet chopped forage from the experimental boxes was immediately compressed into a polyvinyl mini silo (15 cm diameter by 50 cm long cylinder) that was stopper at both ends and contained a valve to allow fermentation gas release (Sheaffer et al. 2006; Hashemzadeh-Cigari et al. 2014). The artificial silos were stored indoors at room temperature. The mini-silos were opened at date of 12/11/2014 and the enough samples was taken to measure pH according to and the rest was dried at 50 ˚C for 48 for other measurements (Hashemzadeh-Cigari et al. 2014).

 

Corn grain collection

Plant from reminded six boxes allowed yielding corn grain. At date of 21/09/2014, corn grain was harvested and weighed and was oven dried at 50 ˚C for 48 h. Weight after drying was recorded to obtain grain biomass yield.

 

Chemical composition analysis

Standard procedures described by the Association of Official Analytical Chemists (AOAC, 1990) were used to determine DM (method 930.15), ash (AOAC method 942.05), crude protein (CP; AOAC method 984.13) and ether extract (EE; AOAC method 954.02). Neutral detergent fiber (NDF), assayed with heat stable alpha-amylase, and acid detergent fiber (ADF) were determined according to the methods for analysis of dietary fiber, neutral detergent fiber, and non-starch polysaccharides in relation to animal nutrition (Van Soest et al. 1991) with the ANKOM A200 Filter Bag Technique (Ankom Technology, Fairport, NY, USA). Acid detergent lignin (ADL) was determined by soaking the ADF filter bag residue in 72% sulphuric acid for 3 h followed by washes with water (AOAC method 973.18). All chemical analyses were performed in duplicate and repeated if required. Hemicellulose (NDF–ADF), cellulose (ADF–ADL), non-fiber carbohydrates, NFC= 100 - (NDF+CP+EE+ash) and total carbohydrates CHO= 100 - (CP+EE+ash) were calculated according to NRC (2001).

 

FTIR scanning of samples

The IR absorbance band of samples was determined using a FTIR spectroscopy (Bruker Tensor 27, Bruker Optics Inc., Billerica, MA, USA) coupled with a universal attenuated total reflectance accessory at the Isfahan University of Technology. The samples were finely ground and pressed uniformly against the diamond surface using a spring-loaded anvil, and the mid-IR spectra recorded from a resolution of 4000 to 600/cm at 2/cm. Typical spectral from corn grain and silages samples in the current study and absorbance bands of interest are shown in Figures 1 and 2 respectively. Each sample was scanned 2 times. Regions of IR spectra identified related to proteins (Amid I + Amid II; 1483 to 1708 cm-1), total carbohydrate (1186 to 852 cm-1), structural carbohydrate (1482.9 to 1188.9 cm-1) and lipids (2770 to 3000 cm-1) (Yari et al. 2013). Amide I and amide II bands are two major bands of the protein infrared spectrum. The amide I band (between 1600 and 1700 cm-1) is mainly associated with the C=O stretching vibration (70-85%) and is directly related to the backbone conformation. Amide II results from the N-H bending vibration (40-60%) and from the C-N stretching vibration (18-40%; Yu, 2005). Functional spectral bands associated with carbohydrate and lipid molecular structures were assigned according to published studies (Refat et al. 2017; Yari et al. 2017) and identified after OriginPro (2019) noise elimination. Functional groups related to molecular structures of total carbohydrates (peak area baseline ca. 852 to 1186 cm-1), structural carbohydrates mainly associated with hemi and cellulosic compounds (peak area baseline ca. 1188 to 1482 cm-1), and lignin (peak area and height, baseline ca. 1498-1526 cm-1) were analyzed (Refat et al. 2017; Yari et al. 2017). Functional groups related to lipids detected from ca 2770 to 3000 cm-1 and are related to vibration absorption of CH (asymmetric stretching of CH2at ca. 2923 cm-1and CH3at ca. 2967 cm-1; CH3 symmetric and CH2 symmetric groups were at ca. 2894 cm-1 and ca. 2874 cm-1 respectively) (Yari et al. 2017). These features were identified after using second derivative and Fourier self-deconvolution functions of OriginPro (2019) (Figure 2 A and B) (Yari et al. 2017). Lipid unsaturation group (CH attached to C=C) peak area is detected between ca. 2996-3020 cm-1 with the center of the peak located at ca. 3000 cm-1 (Yari et al. 2017).

 

In situ ruminal degradability

In situ ruminal incubations were performed as described by (Ørskov and McDonald, 1979) using 2 ruminal fistulated Mehraban ram lambs (body weight=35.5±2.5 kg) fed 1 kg DM/day (in g/kg DM; total mixed ration with 120 g chopped alfalfa hay, 540 g chopped wheat straw, 70 g wheat bran, 80 g ground barley, 170 g beet pulp, 10 g mineral-vitamin supplement, 5 g NaCl and 5 g bentonite) twice daily in equal portions. Feeding and animal husbandry of the lambs were according to procedures by the Iranian Council on Animal Care (1995) guidelines. Corn grain and corn silages samples were pooled in to two samples. Approximately 2 g of each sample was placed in Dacron bags (5 cm×10 cm and pore size of 40 micron). For corn silages, the bags number for incubation interval of 0, 3, 6, 12, 24, 48 and 72 h were 2, 2, 2, 2, 4, 6 and 8. For corn grain samples, ruminal incubation intervals were 0, 24 and 48 h. for both of grain and forage samples, bags for time 0 were manually washed under tap water. For other times, immediately after retrieval from the rumen, bags were manually washed and oven-dried at 50 ˚C for 48 h. The residues in the bags were analyzed for DM and ash as mentioned previously. Rumen degradation parameters of DM and organic matter (OM) were estimated according to the Orskov and McDonald (1979), Y= a + b (1–e-ct), modified first order kinetics model. Parameters were calculated using the nonlinear (NLIN) procedure of SAS (2015) and iterative least-squares regression (Gauss−Newton method). Parameters were Y, the amount of degradable DM and OM at time t of incubation, washable degradation component (a), slowly degradable component (b) and the constant rate of degradation (kd). Effective rumen degradation (ed) was calculated using rate of passage (kp) of 5%. The potential of degradation (pd) was calculated as summation of a and b parameters.

 

In vitro gas production

A semi- automated system was used for in vitro gas production incubations as described by (Theodorou et al. 1994), using buffered rumen fluid prepared according to (Menke and Steinglass, 1988) for all treatments including grain and silage samples. Rumen fluid was collected before the morning feeding from 2 ruminal fistulated ram lambs (same animals as previously mentioned). After collection, ruminal contents were strained through 4 layers of cheese cloth, to eliminate large feed particles, and then transported to the laboratory in a pre-warmed thermal flasks. Three bottles (125 mL) used as laboratory replicates for each sample that were filled with 0.2 g of feed, 10 mL of rumen liquid and 20 mL of buffer (Theodorou et al. 1994) and sealed and incubated for 72 h at 37.5 ˚C. Three bottles with buffered rumen medium, without sample, were incubated to correct for gas release from the inoculum. Head-space gas accumulation were measured using a pressure transducer (Razi Instruments, Mashhad, Iran) and head-space gas volume (Gp) was predicted by Boyle's Gas Law from pressure measurements as:

GP= (Vh/Pa) × Pt

Where:

Vh: head-space volume (95 mL).

Pa: atmospheric pressure (101298.77 Pa; Meteorological Office, Hamadan, Iran).

Pt: represents pressure transducer reading (Theodorou et al. 1994).

Head-space pressure readings were taken at 0, 3, 6, 12, 24, 48 and 72 after the start of incubation. All incubations were repeated into 2 runs. The rate and extent of gas production were determined for each vial by fitting gas production data over time to the nonlinear equation:

Y= b[1 – e -ct]

Where:

Y: volume of gas produced at time t.

b: asymptotic gas production.

c: fractional rate of gas production (Ørskov and McDonald, 1979).

Parameters b and c were calculated using the NLIN (nonlinear) procedure of SAS using iterative least-squares regression (Guass-Newton method; SAS, 2015).

 

Statistical analysis

Data of forage yield, chemical composition, in situ DM and OM degradability, in vitro gas production kinetics were analyzed using PROC MIXED of SAS 9.4 (SAS, 2015) with the following statistical models:

Yij= μ + Ti + Bj + eij

Where:

Yij: observation of the dependent variable ij.

μ: fixed effect of population mean for the variable.

Ti: fixed effect of treatments (i=2; control and P. indica). Bj: random effect of pooled block (j=2).

eij: random error associated with the observation ij.

The Fisher’s protected least significant difference test was used for multiple treatment comparisons using the LSMEAN statement of SAS (2015). For the different statistical tests, significance was declared at P ≤ 0.05 and trends were considered at P ≤ 0.10. The FTIR spectra per corn grain and silage samples were subjected to multivariate and univariate analysis of variation (MANOVA and ANOVA, respectively). The MANOVA analysis was performed using cluster analysis principle component analysis (PCA) by OriginPro (2019). For the ANOVA, firstly the FTIR peak height and area for each functional group was measured by OriginPro (2019) and then by above statistical model the effect of treatment was considered (Yari et al. 2013; Refat et al. 2017).

 

RESULTS AND DISCUSSION

Corn seed samples had similar DM content at time of harvest while samples inoculated with P. indica had higher biomass yield (P=0.05). Samples had similar chemical composition except for total carbohydrate content which tended to be higher for control group (Table 1). In corn forages, DM content of samples and biomass yield at harvest time was similar between treatments (Table 1). Leaf to stem ratio decreased in forage from treated corn (P=0.04) compared with control corn forage. Samples had similar chemical composition except for ether extract which was higher in treated samples with P. indica (P=0.04). In forage samples, in situ ruminal degradability of DM and OM including washable fraction, slowly degradable fraction, fractional rate of degradation, potential of degradation and effective degradability from both experimental groups were similar (Table 2).

 

Figure 1 Full spectrum FTIR from ca 4000 to 600/cm of corn silages from control group and treated corn seedlings with Priformospora indica

Regions of IR spectra identified related to proteins (Amid I+Amid II; 1483 to 1708 cm-1), total carbohydrate (1186 to 852 cm-1), structural carbohydrate (1482.9 to 1188.9 cm-1), lipids (2770 to 3000 cm-1) and lignin (1498-1526 cm-1)

 

 

Figure 2 Full spectrum FTIR from ca 4000 to 600/cm of corn silages from control group and treated corn seedlings with Priformospora indica

Regions of IR spectra identified related to proteins (Amid I+Amid II; 1483 to 1708 cm-1), total carbohydrate (1186 to 852 cm-1), structural carbohydrate (1482.9 to 1188.9 cm-1), lipids (2770 to 3000 cm-1) and lignin (1498-1526 cm-1)

 

 

Table 1 Effect of inoculation of corn seedlings with Priformospora indica on botanical traits and chemical composition of grain and forage harvested as silage

 

1 Botanical traits were measured on fresh forages.

DM: dry matter; OM: organic matter; EE: ether extract; CP: crude protein; NDF: neutral detergent fiber; ADF: acid detergent fiber; ADL: acid detergent lignin; CHO: total carbohydrate calculated according the NRC (2001) as: CHO= 100 - (CP+EE+Ash); NFC: non-fiber carbohydrate calculated according the NRC (2001) as: NFC= 100 -(NDF+CP+Ash+EE).

SEM: standard error of the means.

 

Table 2 In situ ruminal degradability of dry matter and organic matter of corn silages from inoculated and non-inoculated corn seedlings with Priformospora indica

 

a: washable fraction; b: slowly degradable fraction; c: rate of ruminal degradation; pd: potential of ruminal degradation and ed: effective ruminal degradation.

 

In corn seed inoculated with P. indica, in situ ruminal DM and OM degradability at 24 h of ruminal incubation were higher compared with control corn seed (Table 3; P=0.02). In vitro rate of gas production and volume of gas produced until 24 h of incubation were higher for silage from treated corn compared with silage from control corn (P=0.02 and P=0.06 respectively). In vitro gas production kinetics were similar between corn seed samples (Table 4). The FTIR height and area for functional groups of nutrient from control and treated corn with P. indica were similar (Tables 5 and 6, respectively) and for corn silages in Tables 7 and 8. The ratio between amid I to amid II and amid II to lignin area tended to decrease in corn silage from treated corn with P. indica compared with control group (P=0.09; Table 7). Other FTIR height and area and ratios related to functional group in corn and treated corn with P. indica were similar (Tables 7 and 8). The PCA did not discriminate between corn seed samples from control group and P. indica group at the FTIR area related to proteins and lipids but PCA discriminated the FTIR area related to carbohydrate between control group and P. indica group samples (Figure 3; a, b and c). The FTIR area related to proteins, lipids, carbohydrates and structural carbohydrates was not discriminated by PCA between corn silages samples from both experimental groups (Figure 4; a, b, c and d).

 

Grain 

The nutrient contents of corn grain samples and corn silage samples including chemical composition, in situ ruminal degradability of DM and OM, in vitro gas production was consistent with those reported by NRC (2001).

 

Table 3 In situ ruminal degradation at different time of incubations of dry matter and organic matter of corn from inoculated and non-inoculated corn seedlings with Priformospora indica

 

SEM: standard error of the means.

 

Table 4 In vitro gas production fermentation kinetics of silage and grain from inoculated and non-inoculated corn seedlings with Priformospora indica

 

b: potential of gas production; c: rate of gas production and Gas 24: cumulative gas production measurement after 24 h of incubation.

SEM: standard error of the means.

 

Table 5 Effect of inoculation of corn seedlings with Priformospora indica on the ANOVA of the FTIR area of functional groups of corn grain samples

 

CHO: total carbohydrates (1186 to 852 cm-1), total proteins (Amid I+Amid II; 1483 to 1708 cm-1), structural carbohydrate (1482.9 to 1188.9 cm-1), total lipid (2770 to 3000 cm-1), and lignin (ca. 1498-1526 cm-1); CH2s: symmetric stretching of CH2 groups was ca. 2874 cm-1; CH2a: asymmetric stretching of CH2 at ca. 2923 cm-1; AmidI_starch: ratio between amidI to starch; AmidII_starch: ratio between amid II and starch; Lipid_CHO: ratio between lipid and total carbohydrates; Lipid_starch: ratio between lipid and starch; Pro_lipid: ratio between protein and lipid and Amide i_ii: ratio between amid I and amid II.

SEM: standard error of the means.

 

In grain samples, chemical composition were similar but total carbohydrate was grater in samples from P. indica-treated corn. This chemical composition was calculated based on NRC (2001) by summation equations. This finding was in accordance with PCA from total carbohydrates of FTIR area which was differentiated between control group corn and treated corn with P. indica. This may indicate that P. indica increased the magnitude of carbohydrate synthesis. This is also consistent with the increased biomass yield in grain production which was higher for P. indica group. Because most of the corn grain is consisted of carbohydrate (Khan et al. 2011; Macome et al. 2017), therefore it seems that the P. indica increased the synthesis of carbohydrates in grain which resulted to greater grain production. In situ ruminal degradation amount of DM and OM was greater for P. indica group compared with control group. In corn varieties, magnitude of ruminal degradation is mostly determined by the starch quantity and quality (Khan et al. 2011; Macome et al. 2017).

 

Table 6 Effect of inoculation of corn seedlings with Priformospora indica on the ANOVA of the FTIR height of functional groups of corn grain samples

 

CHO: total carbohydrates (1186 to 852 cm-1), total proteins (Amid I+Amid II; 1483 to 1708 cm-1), structural carbohydrate (1482.9 to 1188.9 cm-1); CH2s: symmetric stretching of CH2 groups was ca. 2874 cm-1; CH2a: asymmetric stretching of CH2 at ca. 2923 cm-1; Starch_amidi: ratio between amid I to starch; Starch_amidii: ratio between starch and amid II; Cell_amidi: ratio between cellulosic compounds and amid I; Cell_amidii: ratio between cellulosic compounds and amid II; Amid I_amid II: ratio between amid I and amid II; Starch_cell: ratio between starch and cellulosic compounds; Chs_amidii: ratio between CH2s and amidii; Chs_amidi: ratio between CH2s and amidi; Cha_amidii: ratio between CH2a and amidii; Cha_amidi: ratio between CH2a and amidi; Starch_chs: ratio between starch and CH2s; Starch_cha: ratio between starch and CH2a; Cell_chs: ratio between cellulosic compounds and CH2s and Cell_cha: ratio between cellulosic compounds and CH2a.

 

Current results indicated that the endophytic fungus P. indica affected the FTIR molecular structures of carbohydrate which resulted in higher total carbohydrate content and ruminal in situ degradation content. However, these changes did not affect fermentation profiles of samples in gas production medium. This might be due to the differences between in situ and in vitro methods for feed evaluation (Krizsan et al. 2012). The silage samples from P. indica group had higher EE content. According to Jafari et al. (2018) co-inoculation of Sinorhizobium meliloti and P. indica increased the EE content compared with P. indica- inoculated alfalfa hay alone. The higher EE content may be due to higher absorption of soil nutrient by corn roots as a result of symbiosis with these endophytic microorganisms. The P. indica has been reported to stimulate the phosphorous and water absorption (Ghaffari et al. 2016), and the synthesis of ATP, cell walls and phospholipids in plants were previously found to increase with increasing phosphorous absorption (Kristek et al. 2005). Inoculation or co-inoculation of alfalfa seedlings had no effect on forage yield compared with un-inoculated alfalfa seedlings in the study of (Jafari et al. 2018) while inoculation of barley seedlings with P. indica was found to increase biomass yield (Ghabooli et al. 2013; Ghabooli, 2014). These authors indicated that P. indica changed some biochemical pathways in the plant which promoted higher biomass yield. The lack of response in forage yield in the current study may be due to different corn variety or growth conditions. Many studies have reported that P. indica improved the biomass yield of plants grown under environmental stress condition (Waller et al. 2005; Ghabooli et al. 2013; Murphy et al. 2014). Under normal growing conditions, the effect of plant inoculations with P. indica on forage biomass yield may be negligible or negative depending on plant species (Augé, 2001; Nadeem et al. 2014).

 

Forage

Forages from P. indica group had smaller leaf/stem ratio. Hosseini et al. (2018) reported that P. indica increased the leaf area and root volume. In corn forage the stem is consisted of mainly of structural carbohydrates such as cellulose, hemicellulose and lignin (Jung and Allen, 1995). However, the NDF, ADF and ADL components which measure the cell wall composition were similar between forage samples. Also, the FTIR functional groups related to cell wall components were similar between forage samples. It has been found that lignin (ADL) is essentially indigestible by most ruminant animals and believed to play a major role in the magnitude of digestion of plant by rumen microbes (Jung and Allen, 1995).

 

Table 7 Effect of inoculation of corn seedlings with Priformospora indica on the ANOVA of the FTIR area of functional groups of forage harvested as silage

 

CHO: total carbohydrates (1186 to 852 cm-1), total proteins (Amid I+Amid II; 1483 to 1708 cm-1), structural carbohydrate (1482.9 to 1188.9 cm-1); CH2s: symmetric stretching of CH2 groups was ca. 2874 cm-1; CH2a: asymmetric stretching of CH2 at ca. 2923 cm-1; Starch_amidi: ratio between amid I to starch; Starch_amidii: ratio between starch and amid II; Cell_amidi: ratio between cellulosic compounds and amid I; Cell_amidii: ratio between cellulosic compounds and amid II; Amid I_amid II: ratio between amid I and amid II; Starch_cell: ratio between starch and cellulosic compounds; Chs_amidii: ratio between CH2s and amidii; Chs_amidi: ratio between CH2s and amidi; Cha_amidii: ratio between CH2a and amidii; Cha_amidi: ratio between CH2a and amidi; Starch_chs: ratio between starch and CH2s; Starch_cha: ratio between starch and CH2a; Cell_chs: ratio between cellulosic compounds and CH2s and Cell_cha: ratio between cellulosic compounds and CH2a.

 

Table 8 Effect of inoculation of corn seedlings with Priformospora indica on the ANOVA of the FTIR height of functional groups of forage harvested as silage

 

CHO: total carbohydrates (1186 to 852 cm-1), total proteins (Amid I+Amid II; 1483 to 1708 cm-1), structural carbohydrate (1482.9 to 1188.9 cm-1); CH2s: symmetric stretching of CH2 groups was ca. 2874 cm-1; CH2a: asymmetric stretching of CH2 at ca. 2923 cm-1; Starch_amidi: ratio between amid I to starch; Starch_amidii: ratio between starch and amid II; Cell_amidi: ratio between cellulosic compounds and amid I; Cell_amidii: ratio between cellulosic compounds and amid II; Amid I_amid II: ratio between amid I and amid II; Starch_cell: ratio between starch and cellulosic compounds; Chs_amidii: ratio between CH2s and amidii; Chs_amidi: ratio between CH2s and amidi; Cha_amidii: ratio between CH2a and amidii; Cha_amidi: ratio between CH2a and amidi; Starch_chs: ratio between starch and CH2s; Starch_cha: ratio between starch and CH2a; Cell_chs: ratio between cellulosic compounds and CH2s and Cell_cha: ratio between cellulosic compounds and CH2a.

 

Figure 3 Multivariate PCA spectral analyses for grains from control group (code C) and treated corn seedlings with Priformospora indica (code Pi)

a:total proteins (Amid I+Amid II; 1483 to 1708/cm); b: total carbohydrates (1186 to 852/cm) and c: total lipid (2770 to 3000/cm)

 

 

Figure 4 Multivariate PCA spectral analyses for silages of control group (code C) and treated corn seedlings with Priformospora indica (code Pi)

a: total proteins (Amid I+Amid II; 1483 to 1708 /cm); b: total carbohydrates (1186 to 852/cm); C: structural carbohydrate (1482.9 to 1188.9/cm) and d: total lipid (2770 to 3000/cm)

 

 

This was reflected in in situ ruminal DM and OM degradability in which the parameters for kinetics were similar between both silage samples. However, in silages from P. indica group, rate of gas production was greater than silage from control group. The ADF and NDF component may be similar between two forages while their constitutions such as the cellulose, hemicellulose and properties related to them might be different (Van Soest et al. 1991; Krizsan et al. 2012). The nutritional value of corn silage largely depends on the content and degradability of the starch (Macome et al. 2017). The starch content, as well as the virtuousness of corn kernels, increases with maturity, and the fractional rate of ruminal starch degradation of corn decreases with maturity (Philippeau and Michalet-Doreau, 1997). It may be that the content and degradability of starch of corn kernel changed by P. indica in which influenced the fractional rate of gas production from corn silage. However, the results from gas production and in situ ruminal degradability did not show the same behavior in the case of rate of degradation and fermentation which may be due to the in vitro or in situ parameters which affect the obtained results (Macome et al. 2017). In the study of Hosseini et al. (2018), in comparison with non-inoculated maize, inoculated maize showed lower catalase and ascorbate peroxidase activities which means that they experienced less oxidative stress induced by stressful conditions.

 

CONCLUSION

Inoculation of corn seeds with P. indica increased the biomass yield, mainly changed the FTIR carbohydrates structures and increased the in situ ruminal degradable component. Corn silages from P. indica-inoculated corn seeds had higher ether extract content and higher in vitro fermentation rate. This promises to create a new platform for exploitation of this endophytic fungus in improving the feeding value of corn seeds and silages under semi-arid climate condition.

 

ACKNOWLEDGEMENT

The authors thank Mr Ruzbahani and the staff of Honarestan  Keshavarzi Samen, Malayer for caring, keeping and feeding sheep to collect rumen liquid and in situ trial. Also the authors thank the Isfahan University of Technology central lab for FTIR analysis.

AOAC. (1990). Official Methods of Analysis. Vol. I. 15th Ed. Association of Official Analytical Chemists, Arlington, VA, USA.
Augé R.M. (2001). Water relations, drought and vesicular-arbuscular mycorrhizal symbiosis. Mycorrhiza. 11(1), 3-42.
Chapin Iii F.S., Zavaleta E.S., Eviner V.T., Naylor R.L., Vitousek P.M., Reynolds H.L., Hooper D.U., Lavorel S., Sala O.E. and Hobbie S.E. (2000). Consequences of changing biodiversity. Nature. 405(6783), 234-241.
Ghabooli M. (2014). Effect of Piriformospora indica inoculation on some physiological traits of barley (Hordeum vulgare) under salt stress. Chem. Nat. Comp. 50(6), 1082-1087.
Ghabooli M., Khatabi B., Ahmadi F.S., Sepehri M., Mirzaei M., Amirkhani A., Jorrín-Novo J.V. and Salekdeh G.H. (2013). Proteomics study reveals the molecular mechanisms underlying water stress tolerance induced by Piriformospora indica in barley. J. Proteomics. 94, 289-301.
Ghaffari M.R., Ghabooli M., Khatabi B., Hajirezaei M.R., Schweizer P. and Salekdeh G.H. (2016). Metabolic and transcriptional response of central metabolism affected by root endophytic fungus Piriformospora indica under salinity in barley. Plant Mol. Biol. 90(6), 699-717.
Hashemzadeh-Cigari F., Khorvash M., Ghorbani G., Ghasemi E., Taghizadeh A., Kargar S. and Yang W. (2014). Interactive effects of molasses by homofermentative and heterofermentative inoculants on fermentation quality, nitrogen fractionation, nutritive value and aerobic stability of wilted alfalfa (Medicago sativa) silage. J. Anim. Physiol. Anim. Nutr. 98(2), 290-299.
Hosseini F., Mosaddeghi M.R., Dexter A.R. and Sepehri M. (2018). Maize water status and physiological traits as affected by root endophytic fungus Piriformospora indica under combined drought and mechanical stresses. Planta. 247(5), 1229-1245.
Iranian Council of Animal Care. (1995). Guide to the Care and Use of Experimental Animals. Isfahan University of Technology, Isfahan, Iran.
Jafari M., Yari M., Ghabooli M., Sepehri M., Ghasemi E. and Jonker A. (2018). Inoculation and co-inoculation of alfalfa seedlings with root growth promoting microorganisms (Piriformospora indica, Glomus intraradices and Sinorhizobium meliloti) affect molecular structures, nutrient profiles and availability of hay for ruminants. Anim. Nutr. 4(1), 90-99.
Jung H. and Allen M. (1995). Characteristics of plant cell walls affecting intake and digestibility of forages by ruminants. J. Anim. Sci. 73(9), 2774-2790.
Khan N.A., Cone J.W., Pellikaan W.F., Khan M.A., Struik P.C. and Hendriks W.H. (2011). Changes in fatty acid content and composition in silage maize during grain filling. J. Sci. Food Agric. 91(6), 1041-1049.
Kristek S., Kristek A. and Pavlovic H. (2005). The influence of mycorrhizal fungi (Glomus sp.) on field pea plant survival and growth in drought caused stress conditions. Plant Soil Environ. 51(9), 385-392.
Krizsan S., Nyholm L., Nousiainen J., Südekum K.H. and Huhtanen P. (2012). Comparison of in vitro and in situ methods in evaluation of forage digestibility in ruminants. J. Anim. Sci. 90(9), 3162-3173.
Kumar M., Yadav V., Tuteja N. and Johri A.K. (2009). Antioxidant enzyme activities in maize plants colonized with Piriformospora indica. Microbiology. 155(3), 780-790.
Macome F., Pellikaan W.F., Hendriks W., Dijkstra J., Hatew B., Schonewille J. and Cone J.W. (2017). In vitro gas and methane production of silages from whole-plant corn harvested at 4 different stages of maturity and a comparison with in vivo methane production. J. Dairy Sci. 100(11), 8895-8905.
Mannion A. and Morse S. (2013). A review of Agronomic, Environmental and Socio-Economic Impacts. Centre for Environmental Strategy, University of Surrey, United Kingdom.
Menke K.H. and Steingass H. (1988). Estimation of the energetic feed value obtained from chemical analysis and in vitro gas production using rumen fluid. Anim. Res. Dev. 28, 7-55.
Murphy B.R., Doohan F.M. and Hodkinson T.R. (2014). Yield increase induced by the fungal root endophyte Piriformospora indica in barley grown at low temperature is nutrient limited. Symbiosis. 62(1), 29-39.
Nadeem S.M., Ahmad M., Zahir Z.A., Javaid A. and Ashraf M. (2014). The role of mycorrhizae and plant growth promoting rhizobacteria (PGPR) in improving crop productivity under stressful environments. Biotechnol. Adv. 32(2), 429-448.
Newman E. and Reddell P. (1987). The distribution of mycorrhizas among families of vascular plants. New Phytol. 106(4), 745-751.
NRC. (2001). Nutrient Requirements of Dairy Cattle. 7th Ed. National Academy Press, Washington, DC., USA.
Origin(Pro). (2019). OriginLab Corporation, Northampton, Massachusetts, USA.
Ørskov E. and McDonald I. (1979). The estimation of protein degradability in the rumen from incubation measurements weighted according to rate of passage. J. Agric. Sci. 92(2), 499-503.
Parmesan C. and Yohe G. (2003). A globally coherent fingerprint of climate change impacts across natural systems. Nature. 421(6918), 37-42.
Peškan-Berghöfer T., Shahollari B., Giong P.H., Hehl S., Markert C., Blanke V., Kost G., Varma A. and Oelmüller R. (2004). Association of Piriformospora indica with Arabidopsis thaliana roots represents a novel system to study beneficial plant–microbe interactions and involves early plant protein modifications in the endoplasmic reticulum and at the plasma membrane. Physiol. Plant. 122(4), 465-477.
Pham G.H., Kumari R., Singh A., Malla R., Prasad R., Sachdev M., Kaldorf M., Buscot F., OelmŘller R. and Hampp R. (2008). Axenic culture of symbiotic fungus Piriformospora indica. Plant Surf. Microbiol. 1, 593-613.
Philippeau C. and Michalet-Doreau B. (1997). Influence of genotype and stage of maturity of maize on rate of ruminal starch degradation. Anim. Feed Sci. Technol. 68(1), 25-35.
Refat B., Prates L.L., Khan N.A., Lei Y., Christensen D.A., McKinnon J.J. and Yu P. (2017). Physiochemical characteristics and molecular structures for digestible carbohydrates of silages. J. Agric. Food Chem. 65(41), 8979-8991.
SAS Institute. (2015). SAS®/STAT Software, Release 9.4. SAS Institute, Inc., Cary, NC. USA.
Sheaffer C.C., Halgerson J. and Jung H. (2006). Hybrid and N fertilization affect corn silage yield and quality. J. Agron. Crop Sci. 192(4), 278-283.
Theodorou M.K., Williams B.A., Dhanoa M.S., McAllan A.B. and France J. (1994). A simple gas production method using a pressure transducer to determine the fermentation kinetics of ruminant feeds. Anim. Feed Sci. Technol. 48(3), 185-197.
Van Soest P.V., Robertson J. and Lewis B. (1991). Methods for dietary fiber, neutral detergent fiber, and nonstarch polysaccharides in relation to animal nutrition. J. Dairy Sci. 74(10), 3583-3597.
Varma A., Verma S., Sahay N., Bütehorn B. and Franken P. (1999). Piriformospora indica, a cultivable plant-growth-promoting root endophyte. Appl. Environ. Microbiol. 65(6), 2741-2744.
Verma S., Varma A., Rexer K.H., Hassel A., Kost G., Sarbhoy A., Bisen P., Bütehorn B. and Franken P. (1998). Piriformospora indica, gen. et sp. nov., a new root-colonizing fungus. Mycologia. 90(5), 896-903.
Waller F., Achatz B., Baltruschat H., Fodor J., Becker K., Fischer M., Heier T., Hückelhoven R., Neumann C. and Von Wettstein D. (2005). The endophytic fungus Piriformospora indica reprograms barley to salt-stress tolerance, disease resistance, and higher yield. Proc. Natl. Acad. Sci. US. 102(38), 13386-13391.
Wu M., Wei Q., Xu L., Li H., Oelmüller R. and Zhang W. (2018). Piriformospora indica enhances phosphorus absorption by stimulating acid phosphatase activities and organic acid accumulation in Brassica napus. Plant Soil. 432(1), 333-344.
Yari M., Valizadeh R., Naserian A.A., Jonker A. and Yu P. (2013). Protein molecular structures in alfalfa hay cut at three stages of maturity and in the afternoon and morning and relationship with nutrient availability in ruminants. J. Sci. Food Agric. 93(12), 3072-3080.
Yari M., Valizadeh R., Nnaserian A.A., Jonker A. and Yu P. (2017). Carbohydrate and lipid spectroscopic molecular structures of different alfalfa hay and their relationship with nutrient availability in ruminants. Asian Australasian J. Anim. 30(11), 1575.
Yu P. (2005). Applications of hierarchical cluster analysis (CLA) and principal component analysis (PCA) in feed structure and feed molecular chemistry research, using synchrotron-based fourier transform infrared (FTIR) microspectroscopy. J. Agric. Food Chem. 53, 7115-7127.
Zhang W., Wang J., Xu L., Wang A., Huang L., Du H., Qiu L. and Oelmüller R. (2018). Drought stress responses in maize are diminished by Piriformospora indica. Plant Signal Behav. 13(1), e1414121.