Arbuscular mycorrhizal fungi improve biomass, nutritive value, and digestibility of Opuntia ficus-indica (L.) Miller under different water levels by inducing resistances against cochineal infestation

doi:10.21203/rs.3.rs-3985792/v1

Background: Opuntia ficus-indica (L.) Miller is dominantly growing in arid and semi-arid areas with low nutrient contents. Plants that grow with low-nutrient conditions are more attacked by herbivore insects and develop a strong association with arbuscular mycorrhizal fungi (AMF) to adapt to nutrient, drought, and herbivore insect stress. The objective of this study was to investigate the effects of AMF inoculations and variable soil water levels (SWA) on the biomass, nutrient concentration, nutritional composition, and nutrient digestibility of the spiny and spineless O. ficus-indica by inducing resistance to cochineal stress.

Results: AMF was inoculated to O. ficus-indica plants to improve nutrient value and digestibility by inducing to resist cochineal and drought stresses. Second instar crawlers were exposed to the AMF treated and controlled O. ficus-indica plants. Drought stress reduced the below and above-ground biomass, cladode nutrient content, nutritional composition, and in vitro dry matter digestibility (IVDMD) and in vitro organic matter digestibility (IVOMD). AMF colonization significantly increased biomass production with significant changes in the macro and micro-nutrient concentrations of O. ficus-indica. AMF inoculation significantly increased the IVDMD and IVOMD of both O. ficus-indica types by improving the biomass, organic matter (OM), crude protein (CP), and reduced fiber and ash contents. AMF-inoculated cladodes improved the nutrient concentrations of the cladodes. AMF caused an increase in biomass production, increased tolerance to cochineal stress, and improved nutrient concentration, nutritional composition, and nutrient digestibility performance of O. ficus-indica.

Conclusions: Our study concludes that the role of AMF on the O. ficus-indica plant is not only to resist drought and cochineal stress but also to improve biomass, nutrient concentration, nutritional composition, and nutrient digestibility. The potential of O. ficus-indica to adapt to cochineal stress is controlled by the micro and micro-nutrient concentration performance related to the AMF association.

AMF inoculation

cochineal stress

drought stress

nutrient digestibility

macro and micro-nutrients

The crassulacean acid metabolism (CAM) plants occur in 300 genera belonging to over 16000 species [ ¹ ]. and grouped into three subfamilies, namely Opuntioideae, Pereskioideae, and Cactoideae [ ², ³ ]. Opuntioideae that has 220–350 species [ ³ ] are the largest and differ from all other cactaceae in having glochids and seeds. Opuntia ficus-indica (L.) Miller is an important economic Opuntioideae species with delicious fruits and stems used for food and feed [ ⁴ ]. O. ficus-indica cladodes are cost-effective feed for ruminant animals because of the fast and high land cover, easy and inexpensive to grow, edible, and ability to survive prolonged droughts [ ⁵ ].

Nutrient content, nutritional composition, and digestibility of O. ficus-indica vary with age, cultivar types, species, and fertilization [ ⁶, ⁷ ]. For instance, O. ficus-indica types with few, short, and thin spines are higher in nutrient contents than long and thick spiny types [ ⁶, ⁸ ]. However, spiny, and spineless types of O. ficus-indica are not significantly varied in mineral content and nutritional composition [ ⁹ ]. O. ficus-indica grows in the wild with low fertilizer, no irrigation inputs, and soils with low nutrient contents [ ⁸ ].

Animals found in arid and semi-arid zones get water-dehydration benefits from feeding on cladodes of cactus (Ben Salem et al. 1996) and improve their production performances [ ⁶ ]. Cladodes of the plants are high in carbohydrates, dry matter (DM), starch, and calcium [ ⁹ ]; water, ash, and energy [ ⁵ ]. On the other side, the crude protein (CP), crude fiber (CF), phosphorus (P), and nitrogen (N) content of the plant are low [ ⁹, ⁸, ¹⁰ ]. O. ficus-indica has a low CF, CP, P, and N content and a high-water content, and these limitations can lead to metabolic disorders, low DM intake, nutrient digestibility, diarrhea, and weight loss in animals [ ¹¹, ¹² ].

Proper interventions on cactus growth can improve the nutrition of cactus for animal feed. [ ¹⁰ ] studied the effect of silage making by spineless cladodes Opuntia stricta associated with forages such as Buffel grass (Cenchrus ciliaris L.), Gliricidia (Gliricidia sepium) and Pornunça (Manihot sp.) on improving CF, CP, and N content, DM intake, and DM digestibility of the silage. Cladodes silage making with other forage species had improved intake, digestibility, and nutrient contents. Mixing O. ficus indica with forage legumes can improve the nutrient contents of the feed diets and the silage [ ¹³ ]. O. ficus indica diets mixed with corn maize plants can improve protein stress and improve animal performance [ ¹⁴ ]. Mixing O. ficus indica diets with tef (Eragrostis tef) straw improves nutrient concentration and nutrient composition [ ¹⁵ ]. Interventions employed to improve the nutrient content of O. ficus indica helps to improve the nutrient concentration, nutrient composition, and nutrient digestibility of the diet from the species. However, these studies focused on the improvement of the nutrient content and digestibility of the O. ficus indica plant when mixed with other plant species. O. ficus indica silage-making with protein-rich plants also demands inflated costs in terms of energy, sugar, water, labor, and handling [ ¹¹ ]. AMF technology can independently improve the biomass and availability of nutrient contents in plants [ ¹⁶, ¹⁷ ]. Improving the nutrient content of plants is important to maximize digestive microbial efficiency and stimulating dry matter digestibility [ ¹⁸ ].

AMF is a key factor in promoting the primary productivity of plants [ ¹⁹ ] and nutrient content of woody plants [ ¹⁷, ²⁰ ]. The role of AMF inoculation on the nutrient composition and nutrient digestibility of spiny and spineless O. ficus indica by inducing cochineal infestation resistance was not studied. O. ficus indica growing in arid and semi-arid areas is challenged by cochineal insects [ ²¹, ²² ]. Cochineal sucks the nutrients and water content of O. ficus-Indica plants [ ²³ ] and brings poor nutritional quality of the plant and animal starvation effects [ ²⁴ ]. In arid and semi-arid areas, interactions between plants and AMF play a significant role in inducing plant resistance to insect stresses and in maintaining the nutrient content of plant communities under insect stress [ ²⁵, ²⁵ ]. AMF cochineal interactions in O. ficus-indica plants varied with different environmental factor [ ²² ]. Water stress impact on insect performance is complex and difficult, as water stress may have a positive, negative, or neutral effect on the performance of various herbivore insects [ ²⁶, ²⁷ ].

In our previous study, we concluded that colonization of O. ficus-indica plants with AMF has a major contribution to improved growth, biomass, and gas exchanges of the species [ ²⁰ ]. However, nutrient concentration and nutritional quality improvements of O. ficus-indica through AMF inoculation needs further study. A study of underground processing through AMF could help in nutritional improvement of the O. ficus-indica and its digestible quality in a way that fulfills the need of the livestock since there is a lack of information regarding the AMF technology on growth performance and digestibility of O. ficus-indica cladodes. In this study, we focused on the role of AMF symbiosis in improving the nutrient contents, nutritional values, and digestion of spiny and spineless O. ficus-indica under various soil water conditions (SWA). We hypothesized that: (1) mycorrhizal O. ficus-indica shows higher below and above-ground biomass productions, nutritional content, and composition than without AMF; (2) the nutritional content and composition of O. ficus-indica cladodes increase with an increase in soil water available; (3) mycorrhizal O. ficus-indica shows higher nutritional content, composition, in-vitro digestibility and resistance against cochineal stress than non-mycorrhizal cladodes.

O. ficus-indica plants were cultured in the greenhouse of the Mekelle agricultural research center in Mekelle City, Tigray, Ethiopia (13°29′N, 39°28′E; 2000 m a. s. l.) for nearly two years before cochineal inoculation (from 10-Sep- 2019 to 14-Mar- 2021) and one year after cochineal infestation (from 15-Mar- 2021 to 20-Mar- 2022). The greenhouse had mean day/night temperatures of 26°C/22°C. The mean daily average relative humidity of the greenhouse was 51%.

2.1. Growing of O. ficus-indica plants

We collected one-year-old mother cladodes from the Mekelle research center. The mother cladodes were spiny and spineless. Morphological traits of the cladodes were not significantly varied [ ²⁰ ]. We dried the cladodes under a shaded area for four weeks. We planted one air-dried cladode in upright position in one pot filled with 23.5 kg of autoclaved river pure sand and field soil excavated from the rhizosphere of the O. ficus-indica plantation. The clay, silt, and sand soil textures for the potted soils were 12.8%, 7.1%, and 80.1% respectively. The potted soil had pH 7.77, electrical conductivity 0.11dSm^− 1, organic carbon 1.04%, total nitrogen 0.7%, available potassium 0.5mg/100 g soil, and available phosphorous content 1.103 mg/100 g soil.

2.2. Rhizosphere soil sampling, AMF spore extraction, cultivation, and application

We excavated soil samples from the rhizosphere of O. ficus-indica plantation following [ ²² ] procedures. AMF spore density and AMF genera types were evaluated using the excavated soil samples following [ ²⁸ ] procedures. Potting soil was sieved and autoclaved at 121 ^OC for two hours before inoculation. We cultivated AM fungi in pot cultures with Sorghum bicolor as the host plant. The fractional AMF root colonization and spore density from the AMF-inoculated Sorghum bicolor plants were determined and then the fungal inoculum and microbial wash added to the center of each pot planted with mother cladodes [ ²⁰ ].

2.3. Greenhouse experimental design and treatments

In this study, a three-factorial experiment with two levels of AMF (presence and absence), two O. ficus-indica type (spiny and spineless), and four levels of plant available soil water (SWA), 0–25% of SWA representing T1, 25–50% of SWA representing a T2, 50–75% of SWA representing a T3, and 75–100% of SWA representing a T4 [ ²⁰ ]. We arranged the treatments in a greenhouse workbench in a completely randomized design with seven replications giving a total of 112 O. ficus indica plants. In this study, an additional 12 controlled experimental pots filled with the same amount of mixed soil as the mother cladode planted pots were prepared the determine the exact percent of SWA [ ²⁰ ] and we calculated the amount of water added to the planted pots following [ ²⁹ ].

2.4. Collecting ovipositing females, culturing, and inoculating crawler

We detached ovipositing females from cochineal-infested O. ficus-indica plants using a brush and collected in plastic jars [ ³⁰ ]. The small-size female and male cochineals, crawlers, and dusty waxes were separated from female cochineal by sieving. We cultured the alienated ovipositing females in plastic jars and kept in a shaded area for 72 hours. We produced enough crawlers for the treatments and sixty crawlers inoculated to the two sides of the treated cladodes on March 15/2021 following [ ³¹ ].

2.5. Mycorrhizal root colonization analysis

The presence or absence of arbuscules, vesicles, and hyphae were used to assess mycorrhizal root colonization following the [ ³² ] method. We preserved the root samples collected from the O. ficus-indica plants in plastic jars filled with 50% ethanol [ ³³, ²² ]. We chopped the collected root samples into 1 cm and treated with 10% KHO solution in a heat-resistant jar. The treated roots were autoclaved at 121°C for 15 min [ ²⁸ ]. The roots were bleached and cleaned after washing in 10% H₂O₂ for about 15 minutes, and acidified with 2% HCl for about 1 h at room temperature. We stained the roots in a mix of 0.05% trypan blue (5:1:1 lactic acid: glycerol: distilled water ratio). We washed the stained roots and immersed in 50% glycerol for 1–2 hours, for further de-staining and preservation. Afterward, the magnified gridline intersect method was used to determine the fractional AMF root colonization [ ²⁸, ³² ].

2.6. Biomass production

We uprooted the O. ficus-indica plants from the pots carefully. We carefully removed the roots were from the uprooted plants to determine the below-ground biomass. Total biomass, shoot, and root biomass were determined by harvesting the whole plant. We then calculated both fresh and dry root-to-shoot ratio yields plant per plant. We washed the roots cut from O. ficus-indica thoroughly using water and cleaned with a clean cloth to remove soil and then weighed using a sensitive balance. We recorded individual fresh weights for root and cladodes per plant. The roots and cladodes of O. ficus-indica were oven-dried at 100 ℃ for 24 and 72 hours, respectively, until constant dehydration weight achieved [ ²⁹ ] and dry matter (g/kg fresh cladode of plants) was measured [ ³⁴ ].

\(\text{D}\text{r}\text{y} \text{m}\text{a}\text{t}\text{t}\text{e}\text{r}= \frac{\text{F}\text{r}\text{e}\text{s}\text{h} \text{w}\text{e}\text{i}\text{g}\text{h}\text{t}-\text{D}\text{r}\text{y} \text{w}\text{e}\text{i}\text{g}\text{h}\text{t}}{\text{F}\text{r}\text{e}\text{s}\text{h} \text{w}\text{e}\text{i}\text{g}\text{h}\text{t}} \times 100\) %

2.7. Nutritional composition analysis

Before analysis, the grounded samples passed through a one mm sieve. We weighed three samples per treatment and stored in sealed plastic bottles for laboratory analysis. The ash contents of the samples were determined by taking two grams of milled sub-samples and burned at 550°C for three hours and we calculated the organic matter content by subtracting the percentage of ash from 100 [ ³⁴ ]. The total nitrogen (N) content of all samples was determined by the Kjeldahl procedure. We calculated the crude protein content by multiplying the 6.25 factors with the nitrogen content of the cladodes [ ³⁴ ]. Acid detergent fiber (ADF), acid detergent lignin (ADL), and neutral detergent fiber (NDF) content of samples were determined following the procedures of [ ³⁵ ] by using filter-bag (Ankom® Technology, # F57).

2.8. In vitro digestibility

In-vitro true dry matter digestibility (IVTDMD) and in-vitro true organic matter digestibility (IVTOMD) are laboratory tests used as plant quality indexes for animal feed by animal nutritionists [ ³⁶ ]. IVTDMD and IVTOMD of all samples were determined by ANKOM (2008) Technology -DAISYII incubator using 0.25 g dried samples passed through a one mm sieve size for IVTDMD analysis. The method includes two consecutive digestion phases. Plant materials were incubated under anaerobic conditions with rumen microorganisms for 48 hours at 39°C followed by a 24-hour acid-pepsin digestion phase at 39°C during the first digestion phase. Residual plant materials were collected and oven dried (105°C for 12 hours) after 72-hour incubation.

2.9. Plant nutrient analysis

After calculating the dry weight of the plants, we cut plant samples into pieces using a sharp knife. We washed the cladodes cut from the O. ficus-indica plants thoroughly with distilled water and cleaned with clean clothes to remove dust, cochineal, and cochineal products. The cut plants were dried. We crushed the dry cladode samples with a mortal and packed the resulting powder for the analysis of nutritional concentration, nutritional composition, and in-vitro true dry matter digestibility. Nutritional concentration parameters; phosphorus (P), nitrogen (N), potassium (K), and calcium (Ca) were determined using ultraviolet-visible spectrophotometry analyzer at Mekelle University Laboratory of Geology and total magnesium (Ma), iron (Fe), manganese (Mn), and zinc (Zn) using Atomic absorption spectrometry (AAS) at Ezana Analytical laboratory PLC, Mekelle, Tigray, Ethiopia. A triplicate of approximately five grams of the O. ficus-indica powder placed into a 250 ml conical flask: 5 ml of concentrated H₂SO₄ added following the addition of 25 ml of concentrated HNO₃ and 5 ml of concentrated HCl. We heated the mixtures at 200 ^OC for 1 hour in a fuming hood and then cooled to room temperature. Then, 20 ml of distilled water was added, and we filtered the mixture using filter paper to complete the digestion of organic matter. Lastly, we transferred the mixture to a 50 ml volumetric flask, filled to mark, and let to settle for at least 15 hours. We analyzed Ma, Fe, Mn, and Zn using the resultant supernatant using the [ ³⁷ ].

2.9. Statistical analysis

We analyzed the data for the root and cladode fresh weight, root and cladode dry weight, root/cladode ratio, DM, CP, ADF, NDF, ADL, and ash IVDMD content using the analyses of variance using SPSS 2016 software. Variations of AMF in O. ficus-indica daughter cladodes traits were evaluated using a three-way analysis of variance (ANOVA). Fractional root colonization of AMF in plant roots was evaluated using two-way ANOVA. We performed Gabriel post hoc test for unequal sample size and least significant difference (LSD) for the main effect comparison after checking for the normality of the distribution of the data. We estimated variability using standard errors and we used LSD (α = 0.05) to compare means at P < 0.05.

3.1. Arbuscular Mycorrhizal Fungi Colonization

Water treatment significantly affected the AMF root colonization (Table 1; Fig. 1). Root colonization increased significantly with increased drought stress. AMF root colonization was absent in non-AMF plants (Table 1). Presences of spines were not significant sources of variation.

Table 1

**Effect of** **O. ficus-indica** **type, mycorrhiza, and SWA on growth and digestibility of** **O. ficus-indica**
Parameters	Units	Types		AMF		SWA		Types × AMF		Types × SWA		AMF × SWA		Types × AMF × SWA
		F	P	F	P	F	P	F	P	F	P	F	P	F	P
Root fresh mass	G	0.051	0.660	363.136	< 0.001	15.669	< 0.001	23.374	< 0.001	6.590	< 0.001	409.938	< 0.001	28.877	< 0.001
Cladode fresh mass	G	0.132	0.418	393.818	< 0.001	9.254	< 0.001	38.805	< 0.001	4.445	0.001	128.158	< 0.001	24.090	< 0.001
Fresh root: cladode ratio	G	0.733	0.556	6.592	< 0.001	82.679	< 0.001	5.073	0.008	20.111	< 0.001	483.417	< 0.001	32.365	< 0.001
Root dry mass	G	0.279	0.522	134.042	< 0.001	36.121	< 0.001	14.110	< 0.001	14.549	< 0.001	577.165	< 0.001	45.545	< 0.001
Cladodes dry mass	G	0.615	0.583	225.723	< 0.001	11.553	< 0.001	28.681	< 0.001	6.630	< 0.001	260.174	< 0.001	27.898	< 0.001
Dry root: cladode ratio	G	0.506	0.804	0.642	< 0.001	12.551	< 0.001	38.869	< 0.001	232.894	< 0.001	19.049	< 0.001	4.598	< 0.001
Dry matter	G	0.720	0.521	16.654	< 0.001	10.894	< 0.001	3.145	0.053	3.419	0.011	73.238	< 0.001	2.660	0.014
Ash	%	0.135	0.855	11.535	< 0.001	6.653	0.001	13.805	< 0.001	2.783	0.029	36.241	< 0.001	10.423	< 0.001
Organic matter	%	0.135	0.855	11.535	< 0.001	6.653	0.001	13.805	< 0.001	2.783	0.029	36.241	< 0.001	10.423	< 0.001
Crude protein	%	0.006	0.848	114.153	< 0.001	14.099	< 0.001	1.520	0.221	1.096	0.338	282.885	< 0.001	1.520	0.203
Acid detergent fiber	%	0.041	0.801	8.261	< 0.001	16.844	< 0.001	13.498	< 0.001	2.557	0.042	47.338	< 0.001	9.987	< 0.001
Acid detergent lignin	%	0.056	0.875	5.486	< 0.001	4.577	0.007	7.228	0.002	5.768	< 0.001	81.661	< 0.001	14.176	< 0.001
Neutral detergent fiber	%	0.001	0.896	12.246	< 0.001	6.653	0.001	14.521	< 0.001	2.273	0.110	41.722	< 0.001	8.767	< 0.001
IVDMD	%	0.001	0.992	97.982	< 0.001	3.101	0.036	13.643	< 0.001	1.463	0.222	57.161	< 0.001	8.812	< 0.001
IVOMD	%	0.002	0.998	126.590	< 0.001	4.925	0.005	15.884	< 0.001	2.273	0.065	100.324	< 0.001	11.091	< 0.001
Nitrogen concentration	%	0.006	0.848	114.153	< 0.001	14.099	< 0.001	1.520	0.221	1.096	0.338	282.885	< 0.001	1.520	0.203
Phosphorus concentration	%	0.018	0.932	373.042	< 0.001	39.362	< 0.001	15.623	< 0.001	17.710	< 0.001	390.091	< 0.001	7.863	< 0.001
Potassium concentration	%	0.061	0.744	132.525	< 0.001	57.183	< 0.001	1.969	0.164	1.962	0.146	67.495	< 0.001	0.826	0.512
Calcium concentration	%	1.647	0.535	152.230	< 0.001	2.923	0.044	8.037	0.001	2.238	0.068	224.930	< 0.001	9.230	< 0.001
Magnesium concentration	%	0.002	0.993	207.983	< 0.001	3.602	0.021	11.086	< 0.001	1.439	0.230	103.658	< 0.001	7.752	< 0.001
Iron concentration	%	1.527	0.424	153.328	0.001	4.728	0.006	2.013	0.145	2.883	0.025	18.316	< 0.001	6.531	< 0.001
Manganese concentration	%	0.000	0.883	102.302	< 0.001	7.963	< 0.001	4.767	0.013	3.339	0.013	224.126	< 0.001	9.469	< 0.001
Zinc concentration	%	0.281	0.840	183.751	< 0.001	7.064	0.001	3.334	0.045	2.965	0.022	304.750	< 0.001	7.909	< 0.001
Hyphal colonization	%	0.101	0.860	-	-	32.000	< 0.001	-	-	9.625	< 0.001	-	-	39.472	< 0.001
Arbuscular colonization	%	0.156	0.641	-	-	43.708	< 0.001	-	-	10.924	< 0.001	-	-	36.145	< 0.001
Vesicular colonization	%	0.079	0.831	-	-	77.758	< 0.001	-	-	8.830	< 0.001	-	-	24.841	< 0.001
P < 0.05 indicates significant sources of variation between treatments Note: AMF: Arbuscular mycorrhizal fungi, SWA: Soil water available, IVDMD: in vitro dry matter digestibility, IVOMD: in vitro dry matter digestibility

3.2. Biomass production

AMF-inoculation and SWA significantly affected fresh mass, dry mass, and root/cladode ratio of O. ficus-indica (Table 1; Fig. 3). There was a significant interaction among O. ficus-indica type × AMF, O. ficus-indica type × SWA, AMF × SWA, and O. ficus-indica type × AMF × SWA in all biomass parameters. In this study, we observed that the below-ground interaction (O. ficus-indica type × AMF × SWA) affected the below and above-ground biomass of O. ficus-indica plants. Because the main inputs of the biomass of O. ficus-indica per pot is based on the effect of AMF accounts for the plant performance variance attributable to the difference between mycorrhizal and nonmycorrhizal O. ficus-indica plants.

3.3. Nutritional composition

The presence of spines in the cladodes did not bring a significant effect on the nutritional compositions of the O. ficus-indica cladodes (Table 1). AMF and SWA had significant on the nutritional compositions of the plants. Dry matter, organic matter, and crude protein content were significantly higher in AMF O. ficus-indica than non-AMF O. ficus-indica plants. Ash content, ADF, ADL, and NDF were lower in AMF O. ficus-indica plants (Fig. 2). An increase in SWA levels significantly increased the dry matter, organic matter, and crude protein content of the cladodes while decreased the Ash content, ADF, ADL, and NDF. O. ficus-indica type × AMF interactions significantly affected the nutritional composition in cladode ash content, OM, CP, ADF, ADL, and NDF (Table 1). The interactions between SWA and O. ficus-indica type significantly affected the DM, ash, OM, ADF, and ADL of daughter cladodes. O. ficus-indica type × AMF × SWA interaction significantly affected DM, ash, OM, CP, ADF, ADL, and NDF.

3.4. In vitro digestibility

We assessed the effect of O. ficus-indica type, mycorrhiza, and plant-soil water available and their interaction on change in vitro dry matter (IVDMD) and in vitro organic matter digestibility (IVOMD) of O. ficus-indica cladodes after exposed to cochineal attack for twelve months. We found that AMF and SWA significantly affected the digestibility of cladodes (Table 1; Fig. 2). The non-mycorrhizal O. ficus-indica cladodes were lower in both IVDMD and IVOMD. There was significant interaction for O. ficus-indica type × AMF, AMF × SWA, and O. ficus-indica type × AMF × SWA for IVDMD and IVOMD.

3.5. Nutrient contents

The presence of spines in the cladodes did not bring a significant effect on the nutritional compositions of the O. ficus-indica cladodes (Table 1). AMF and SWA had significant on the nutritional compositions of the plants. Dry matter, organic matter, and crude protein content were significantly higher in AMF O. ficus-indica than non-AMF O. ficus-indica plants. Ash content, ADF, ADL, and NDF were lower in AMF O. ficus-indica plants (Fig. 2). An increase in SWA levels significantly increased the dry matter, organic matter, and crude protein content of the cladodes while decreased the Ash content, ADF, ADL, and NDF. O. ficus-indica type × AMF interactions significantly affected the nutritional composition in cladode ash content, OM, CP, ADF, ADL, and NDF (Table 1). The interactions between SWA and O. ficus-indica type significantly affected the DM, ash, OM, ADF, and ADL of daughter cladodes. O. ficus-indica type × AMF × SWA interaction significantly affected DM, ash, OM, CP, ADF, ADL, and NDF.

3.4. In vitro digestibility

We assessed the effect of O. ficus-indica type, mycorrhiza, and plant-soil water available and their interaction on change in vitro dry matter (IVDMD) and in vitro organic matter digestibility (IVOMD) of O. ficus-indica cladodes after exposed to cochineal attack for twelve months. We found that AMF and SWA significantly affected the digestibility of cladodes (Table 1; Fig. 2). The non-mycorrhizal O. ficus-indica cladodes were lower in both IVDMD and IVOMD. There was significant interaction effect for O. ficus-indica type × AMF, AMF × SWA, and O. ficus-indica type × AMF × SWA for IVDMD and IVOMD.

Our study supported the hypothesis that mycorrhizal O. ficus-indica shows higher below and above-ground biomass productions, nutritional content, and composition than without AMF. We observed a positive effect of mycorrhizal symbiosis on the below and above-ground biomass increment of O. ficus-indica under greenhouse conditions using spiny and spineless cladodes inoculated with AMF. Increased below and above-ground biomass in AMF plants compared to non-mycorrhizal ones reported for other species [ ³³, ¹⁷ ]. The greater plant biomass measured from treatments inoculated with AMF could be due to enhanced nutrient uptake due to increased root surface area [ ³⁸ ]. [ ²⁰ ] reported that AMF inoculations enhance the biomass of spiny and spineless O. ficus-indica plants through improved growth, photosynthetic water use efficiency, and photosynthesis. [ ³³ ] reported a positive mycorrhizal effect on the biomass of Boswellia papyrifera seedlings over control seedlings due to significantly improved growth, gas exchange, and P nutrition.

AMF inoculation caused a significant impact on biomass by the resistance of AMF to cochineal insects. Similarly, there are studies on enhanced biomass production of plants under insect stress due to AMF inoculation [ ³⁹, ²⁵, ²² ]. It is important to note that the above-ground biomass of the non-AMF-inoculated plants that was severely destroyed by the cochineal could possibly have been altered by the sucking nutrients of the plant; therefore, we observed a strong difference in macro and micro-nutrients between the AMF-inoculated and controlled plants (Table 1; Fig. 4).

The biomass production significantly reduced with increased drought stress on both spiny and spineless plant types. Reduced biomass production of the plant in response to low levels of SWA has been well documented [ ²⁰ ] and observed in CAM plants [ ⁴⁰, ⁴¹ ]. This reduced biomass production performance is to be due to reductions in overall O. ficus-indica morpho-physiological performance [ ²⁰ ]. However, AMF-inoculated plants were better adapted to drought stress than controlled plants. Similarly, [ ⁴² ] and [ ¹² ] found that AMF-inoculated plants better induce tolerance to drought stress and improve biomass production than non-mycorrhizal plants.

For the first time, to our knowledge, we investigated the impact of AMF inoculation, SWA, and O. ficus-indica type on O. ficus-indica performance under nutrient-sucking cochineal insect stress. AMF inoculations effectively reduced the impact of cochineal on the O. ficus-indica biomass, nutrients, and national performance, while the SWA and types were not related to the cochineal impact. These responses are likely linked to changes in plant nutrients [ ³⁰ ]. However, under cochineal-stressed conditions, the two- and three-way ANOVA interaction results showed that AMF, SWA, and types affected the plant performance measured parameters. In contrast to our observations, drought stress can only negatively affect the performance of western flower thrips, Frankliniella occidentalis (Thysanoptera: Thripidae), on tomatoes [ ⁴³ ].

Our finding supported the hypothesis that the nutritional content and composition of O. ficus-indica cladodes increase with the increase in the amount of soil water available. Nutritional composition decreased with an increase in drought stress (Table 1). These results are consistent with the findings of [ ⁶ ], who investigated the effects of water stress on the chemical composition of spiny (Opuntia amyclae) and spineless (Opuntia ficus indica f. inermis) cactus cladodes. The result of this study showed that OM, CP, ADF, and lignin content were higher for water-stressed plants than for non-stressed plants. However, DM content was higher at non-stressed than water-stressed plants.

As hypothesized, we found higher IVDMD and IVOMD in the mycorrhizal O. ficus-indica cladode types than non-mycorrhizal cladodes with the resistance of drought and cochineal stresses. These results can relate to the significant impact of AMF inoculation on the increment in weight, nutritive value, and nutritional composition (CP and OM) of the plant (Table 1). IVDMD and IVOMD in the O. ficus-indica cladode increased with an increase in biomass, nutritive value, and nutritional composition (CP and OM) and a reduction in fiber content, consistent with the findings of [ ¹⁵ ] of the plant. CAM plans have poor nutrient digestibility due to the low contents of crude protein (CP), crude fiber (CF), phosphorus (P), and nitrogen (N) [ ⁹, ⁸,¹⁰ ]. However, AMF inoculation technology in this study improved the nutrient digestibility of O. ficus-indica and improved all these limited nutrient contents.

The role of AMF on the O. ficus-indica plant is not only to resist drought and cochineal stress but also to improve biomass, nutrient concentration, nutritional composition, and nutrient digestibility. AMF caused an increase in nutrient digestibility, which was related to the improvement of OM, CP, P, and N contents. A decrease in Ash, ADF, ADL, and NDF content clue to improved IVOMD and IVDMD digestibility of the mycorrhizal O. ficus-indica. Cochineal stress brought low biomass production in the non-mycorrhizal plants. AMF-colonized O. ficus-indica plants inoculated with cochineal had better nutrient contents. Cochineal stress was not significantly related to drought stress or O. ficus-indica types. The levels of SWA affect the impact of AMF root colonization performance on O. ficus-indica. AMF has interacted with decreasing levels of SWA. Thus, mycorrhizal O. ficus-indica plants with low levels of SWA increased their benefits for drought stress. AMF associated with the O. ficus-indica type and SWA improved nutrient concentration, nutritional composition, and nutrient digestibility. The presence of a spine on the surface area of O. ficus-indica cladodes cannot cause decreased or increased nutrient concentration, nutritional composition, or nutrient digestibility. AMF O. ficus-indica plants should be colonized by AMF to improve the nutrient concentration, nutritional composition, and nutrient digestibility by resisting cochineal and drought stress.

Acknowledgement

We thank Mekelle University Departments of Land Resource Management and Environmental Protection (LaRMEP) and Geology for their permission to do Laboratory analysis in their respective laboratories. We thank the staff of the Ezana Analytical Laboratory PLC, Mekelle, Tigray, Ethiopia.

Authors’ contributions

T.G.K. and E.B. Conceptualization the idea, and performed the experiments; T.G.K. expresses the data collection and analysis, and writing - original draft manuscript; E. B., K-M.A. and Y.G. provided guidance and supervision during the experiments and manuscript writing. All authors have read and approved the manuscript for publication.

Funding

This work was supported by the Ethiopia Ministry of Education and Institute of International Education-Scholars Rescue Fund (IIE-SRF), the Norwegian University of Life Sciences (NMBU), the Faculty of Environmental Sciences and Natural Resource Management (MINA), and the NORGLOBAL 2 project “Towards a climate-smart policy and management framework for conservation and use of dry forest ecosystem services and resources in Ethiopia [grant number: 303600]” for supporting the research stay of Emiru Birhane at NMBU.

Availability of data and materials

The relevant datasets supporting the results of this article are available with the authors and will include within the article link at respective time for publication process.

Ethics approval and consent to participate

We declare that all the experimental AMF inoculums, cochineal, and plants were collected with permission from local authorities of agricultural office and all experimental studies on plants were complied with relevant institutional, national, and international guidelines and legislation.

Consent for publication

Not applicable.

Conflict of interest

The authors declare no conflict of interest.

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No competing interests reported.

Arbuscular mycorrhizal fungi improve biomass, nutritive value, and digestibility of Opuntia ficus-indica (L.) Miller under different water levels by inducing resistances against cochineal infestation

Status:

Journal Publication

Version 1

Abstract

Figures

Introduction

Materials and methods

2.2. Rhizosphere soil sampling, AMF spore extraction, cultivation, and application

2.3. Greenhouse experimental design and treatments

2.4. Collecting ovipositing females, culturing, and inoculating crawler

2.5. Mycorrhizal root colonization analysis

2.6. Biomass production

2.7. Nutritional composition analysis

2.8. In vitro digestibility

2.9. Plant nutrient analysis

2.9. Statistical analysis

Results

3.1. Arbuscular Mycorrhizal Fungi Colonization

3.2. Biomass production

3.3. Nutritional composition

3.4. In vitro digestibility

3.5. Nutrient contents

3.4. In vitro digestibility

Discussion

Conclusions

Declarations

References

Additional Declarations

Status:

Journal Publication

Version 1