Effects of endophytic and saprophytic fungi on in vitro methanogenesis

Figures & data

Table 1  The species and strains of saprophytic fungi associated with spoilage of silage and selected for screening, their source and some characteristic production of secondary metabolites that may influence enteric methane production.

Fungus	Strain	Source	Characteristics of fungus
Mortierella wolfii	68/707w2	di Menna^1	Produces a protein nephrotoxin, grows at 37 °C and causes mycosis in cattle (Munday et al. 2010: abortion and pneumonia; encephalitis when injected into brain of mice).
	69/3891	di Menna
	74/2649	di Menna
Penicillium roquefortii	A2	di Menna	Regarded as a complex of three species (Boysen et al. 2000: P. roquefortii, P. paneum and P. carneum—the first two being associated with mouldy silage P. carneum mainly with bread). Produces extensive range of mycotoxins including: • Roquefortine (several) • Mycophenolic acid • Eremofortin C • PR toxin • Penitrem A
	G2	di Menna
	I2	di Menna
Monascus ruber	15220	ICMP^2	A red mould used in fermented food products that produces Citrinin (active against G+ bacteria) and Monacolin K = lovastatin (Wolin & Miller 2006: statins may affect methanogenesis, but also growth of anaerobic fungi).
	503.70	CBS^3
	554.76	CBS
Geotrichum candidum	240.62	CBS	May produce clavine alkaloids (Sallam et al. 1970).
^1 Dr Margaret di Menna, AgResearch, Hamilton.
^2 International Collection of Microorganisms from Plants, Landcare Research, Auckland, New Zealand.
^3 Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands.

Table 2  Concentrations of the major classes of alkaloids lolitrem B, ergovaline (Ev), peramine (Per), lolines and epoxy-janthitrems (Epox) in several strains of Neotyphodium lolii endophyte in perennial ryegrass (GA66) or N. coenophialum endophyte in tall fescue (cv Jesup).

		Alkaloid (mg/kg DM)
Species	Endophyte strain	Lolitrem	Ev	Per	Lolines	Epox
P. ryegrass	Wild type	1.7	0.4	22.7	–^1	–
	EL1	1.3	<0.1	–	–	–
	AR6	–	1.1	14.2	–	–
	EL2	–	1.1	9.1	–	–
	EL3	–	0.6	7.7	–	–
	EL4	–	0.2	9.7	–	–
	AR50	–	<0.1	24.3	–	–
	AR1	–	–	21.3	–	–
	AR37	–	–	–	–	21.7
	EL5	–	–	–	–	–
	Endophyte-free	–	–	–	–	–
Tall fescue	Wild type	–	0.2	3.9	1188	–
	AR525	–	–	5.0	687	–
	AR542	–	–	2.5	217	–
	AR584	–	–	4.0	344	–
	EL6	–	–	5.0	419	–
	Endophyte-free	–	0.1	0.4	–	–
^1 None detected

Table 3  Total gas production (GP), methane (CH₄) gas as a proportion of GP, the concentration of total short chain fatty acids (SCFA), and acetate, propionate and butyrate as proportions of SCFA after 24 h in vitro incubation of several strains of Neotyphodium lolii endophyte in perennial ryegrass (GA66) or N. coenophialum endophyte in tall fescue (cv Jesup).

		GP	CH4	SCFA	Acetate	Propionate	Butyrate
Grass	Endophyte strain	ml/g DM	% of GP	mM	%	%	%
P. ryegrass	Endophyte-free	184.1	20.4	41.0	62.5	22.8	10.8
	Wild type	186.2	20.2	41.6	63.2	24.0	9.4
	EL1	182.7	21.6	43.3	62.0	21.4	11.8
	AR6	177.2	20.6	35.9*	58.4	27.6*	11.5
	EL2	183.4	21.6	44.6	61.5	21.4	12.1
	EL3	191.5	20.4	43.0	62.4	24.9*	10.0
	EL4	181.4	20.3	38.4	61.1	24.2	10.8
	AR50	180.8	20.6	42.8	61.5	21.4	12.2
	AR1	175.7	20.6	36.9	63.3	23.5	8.6
	AR37	182.8	20.9	35.6*	63.3	24.3	9.2
	EL5	181.0	20.8	38.3	64.2	24.1	9.0
	Ryegrass mean	182.4	20.7	40.1	62.3	23.6	10.5
	SED	4.60	0.86	2.12	1.55	0.91	1.22
	P value	0.302	0.858	0.013	0.110	<0.001	0.479
Tall fescue	Endophyte-free	141.4	21.5	37.0	63.4	22.6	9.4
	Wild type	164.0*	19.6	48.3*	64.8	22.1	7.8
	AR525	145.8	20.9	37.8	64.3	23.5	7.8
	AR542	172.5*	21.8	46.1*	63.0	23.1	8.4
	AR584	167.9*	20.8	42.5*	62.9	23.2	8.5
	EL6	183.0*	20.4	51.5*	64.0	22.4	8.8
	Tall fescue mean	162.4	21.0	44.2	63.6	22.9	8.5
	SED	6.16	0.99	2.30	1.30	0.60	0.50
	P value	0.002	0.468	0.002	0.700	0.200	0.100
* Indicates grass-endophyte associations that are significantly different from the endophyte-free (negative) control based on Fisher's protected LSD0.05.

Table 4  Total gas production (GP) and methane (CH₄) gas as a proportion of GP after 9 h and 24 h in vitro incubation, and the concentration of total short chain fatty acids (SCFA) and acetate, propionate and butyrate as proportions of total SCFA after 24 h in vitro incubation of negative and positive controls and fungal mat and supernatant fractions of several species and strains of saprophytic fungi.

			Total gas		Methane gas
			ml/g DM		% of GP		SCFA
Fungus			GP	GP	CH4	CH4	Total	Acetate	Propionate	Butyrate
Fraction	Species	Strain	9 h	24 h	9 h	24 h	mM	%	%	%
	Negative control		82.8	153	15.0	17.0	39.7	65.8	22.9	8.3
	Positive control		69.7	133	7.5	12.2	38.8	62.2	25.0	9.9
Fungal mat	Mortierella wolfii	69/3891	75.6*	141.4*	14.6	15.9*	36.4	61.0	27.6*	7.8
	Mortierella wolfii	74/2649	102.7*	180.6*	14.6	16.6	41.6	68.6	20.7*	7.8
	Mortierella wolfii	68/707w2	97.7*	172.5*	14.8	16.9	41.3	68.7	20.6*	7.8
	Penicillium roquefortii	G2	77.0	149.7	13.0*	13.9*	33.7*	58.6	29.4*	8.4
	Penicillium roquefortii	I2	88.7	165.0*	17.3*	19.1*	40.1	66.7	20.6*	9.1
	Penicillium roquefortii	A2	98.5*	174.1*	17.9*	19.2*	41.1	67.3	20.0*	9.0
	Monascus ruber	15220	106.6*	184.0*	17.2*	18.7*	44.3*	66.5	20.4*	9.3
	Monascus ruber	503.7	75.5*	157.9	11.8*	14.0*	34.9*	64.3	23.8	9.0
	Geotrichum candidum	240.62	73.9*	156.9	12.2*	14.3*	33.1*	64.3	23.8	8.8
	Monascus ruber	554.76	72.5*	151.6	11.8*	13.9*	32.4*	63.6	24.5*	9.0
	Fungal mat mean		86.9	163.4	14.5	16.3	38.6	65.2	23.0	8.7
		SED	2.68	5.14	0.55	0.40	1.77	0.93	0.65	0.30
		P value	<0.001	<0.001	<0.001	<0.001	0.032	0.33	0.042	0.153
Supernatant	Mortierella wolfii	69/3891	82.8	155.3	10.0*	11.6*	38.5	61.0	28.5	7.3
	Mortierella wolfii	74/2649	109.8*	185.7*	10.2*	12.2*	46.6*	58.8	30.3	7.9
	Mortierella wolfii	68/707w2	105.3*	185.8*	8.8*	10.6*	44.5*	57.4	30.6	7.7
	Penicillium roquefortii	G2	75.9	142.7	11.1*	13.4*	32.4*	56.4	31.5	8.5
	Penicillium roquefortii	I2	95.2*	172.6	15.6	17.8	43.1*	67.9	20.0	8.6
	Penicillium roquefortii	A2	100.1*	176.0*	15.4	17.9	44.0*	67.5	20.2	8.9
	Monascus ruber	15220	80.6	153.3	14.9	17.5	39.9	68.4	20.1	8.5
	Monascus ruber	503.7	109.7*	197.0*	9.1*	12.4*	45.3*	61.1	26.3	9.9
	Geotrichum candidum	240.62	110.7*	206.0*	8.8*	12.3*	45.1*	61.7	25.9	9.8
	Monascus ruber	554.76	106.7*	194.5*	8.4*	11.9*	45.2*	60.7	26.9	9.8
	Supernatant mean		97.6	176.9	11.2	13.8	42.3	62.7	25.6	8.6
		SED	4.05	9.60	0.86	0.73	1.91	2.23	1.61	0.54
		P value	<0.001	<0.001	<0.001	<0.001	<0.001	0.066	0.062	0.078
* Indicates strains that are significantly different from the negative control based on Fisher's protected LSD0.05.

Table 5  The ranking score of fungal mat and supernatant fractions of several species and strains of saprophyte fungi and the positive control, calculated from the effects on production of methane and short chain fatty acids during in vitro fermentation relative to the negative control (the method of calculation gives the negative control a score of zero).

Fungus	Strain	Fraction	Ranking
Mortierella wolfii	68/707w2	Supernatant	1.08*
Mortierella wolfii	74/2649	Supernatant	0.97*
Mortierella wolfii	69/3891	Supernatant	0.79*
Positive control	–	–	0.77*
Monascus ruber	554.76	Supernatant	0.69*
Geotrichum candidum	240.62	Supernatant	0.63*
Monascus ruber	503.7	Supernatant	0.60*
Penicillium roquefortii	G2	Supernatant	0.46*
Penicillium roquefortii	G2	Fungal mat	0.35*
Mortierella wolfii	69/3891	Fungal mat	0.22*
Penicillium roquefortii	A2	Supernatant	0.20*
Penicillium roquefortii	I2	Supernatant	0.17*
Monascus ruber	15220	Supernatant	0.15
Monascus ruber	15220	Fungal mat	0.07
Negative control	–	–	0.00
Mortierella wolfii	74/2649	Fungal mat	−0.03
Monascus ruber	503.7	Fungal mat	−0.03
Penicillium roquefortii	I2	Fungal mat	−0.06
Mortierella wolfii	68/707w2	Fungal mat	−0.07
Penicillium roquefortii	A2	Fungal mat	−0.08
Monascus ruber	554.76	Fungal mat	−0.10
Geotrichum candidum	240.62	Fungal mat	−0.14
	SED		0.09
	P value		<0.001
* Indicates strains that are significantly different from the negative control based on Fisher's protected LSD0.05.

Boysen , ME , Jacobsson , KG and Schnürer , J . 2000 . Molecular identification of species from the Penicillium roquefortii group associated with spoiled animal feed . Applied and Environmental Microbiology , 66 : 1523 – 1526 .

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Sallam , LAR , El-Refai , AH and Naim , N . 1970 . The alkaloids of fungi. II. Some factors influencing alkaloid formation by Geotrichum candidum . Zeitschrift für allgemeine Mikrobiologie , 10 : 405 – 412 .

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