Enhancing potato crop yields through AMF colonization
Plant-parasitic nematodes pose a significant threat to global food production, causing annual crop losses exceeding USD $80 billion. A notable nematode, the white potato cyst nematode (Globodera pallida), is highly destructive, infiltrating the roots of solanaceous hosts, particularly potatoes, and establishing feeding sites that support its development and reproduction. This nematode is widespread in potato-producing regions and, along with its close relative, Globodera rostochiensis, contributes to nearly 10% of global potato crop losses. Recent findings have detected G. pallida cysts in 43% of UK potato fields, raising concerns in Africa as well.
The multiplication rate of G. pallida is heavily influenced by the resistance of the cultivated potato variety, leading to long-term implications due to the nematode's persistence in agricultural soils, even in the absence of a host. Factors like soil temperature, rainfall, and the cultivation of susceptible or resistant/non-host plants can impact nematode density in fields. Higher densities of G. pallida result in greater yield losses and limit growers' variety choices, forcing them to select options that can tolerate higher nematode burdens.
In fields infected by G. pallida, potato roots are often simultaneously colonized by various soil-borne symbionts, including arbuscular mycorrhizal fungi (AMF). AMF are widespread in soils and form mutualistic relationships with plants, aiding in nutrient uptake. Recent studies have shown that when potato roots are co-colonized by G. pallida and AMF, there is a shift in carbon allocation, with AMF partners receiving less while nutrient transfer to the host remains stable. This presence of AMF in co-colonized plants seems responsible for boosting G. pallida fitness and enhancing host plant tolerance to the nematode.
However, these findings were based on a single density of G. pallida on plant roots. In the field, G. pallida infection densities can vary, influencing resource allocation. High G. pallida densities can significantly impact host plant tolerance. The impact of G. pallida density on AMF-induced tolerance in potatoes remains uncertain. While AMF appear to increase G. pallida populations and improve plant vigor by enhancing nutrient supply, this may not hold under high G. pallida densities, potentially leading to crop failure and persistently high G. pallida populations.
Furthermore, AMF colonization of the host can also increase populations of other plant-parasitic nematodes, expanding the problem. Field trials are exploring the use of AMF as soil amendments to improve yields, enhance the biosynthesis of desirable phytochemicals, and increase plant tolerance to pests and abiotic stresses. This approach offers an alternative to heavy pesticide use, which faces restrictions for environmental reasons. Field trials have shown promising results, particularly in potato cultivation. However, the effectiveness of AMF applications can vary depending on factors like plant and fungal genotypes, compatibility issues, competition with native species, field capacity for AMF, and farming practices.
To date, only a limited number of field trials have investigated the application of AMF inoculum to enhance plant tolerance to plant-parasitic nematodes. Some experiments have shown AMF to reduce infection and enhance host plant tolerance to root-knot nematodes. Field trials with cyst nematodes suggest that AMF can promote hatching and potentially work synergistically with nematicides to reduce nematode populations in the soil. Whether AMF can effectively mitigate the effects of cyst nematode pests in field environments remains a topic requiring further investigation.
In this work, greenhouse and field experiments were conducted to assess the impact of G. pallida density on AMF-induced tolerance and G. pallida fitness. Additionally, we examined the influence of a commercial AMF inoculant on the native AMF community.
All plants were grown in a controlled environment in a containment glasshouse with specific conditions (18–20°C) temperature and 16-hour day length) and received regular watering. G. pallida inoculum was created by infecting plants with Globodera pallida cysts and grown for 12 weeks with regular fertilizer applications until plant death to increase G. pallida populations for later use.
The PCN content of the inoculum soils was determined through Fenwick's (1940) method and mixed with sterilized sand and loam topsoil to achieve desired PCN densities of 15, 35, and 125 eggs per gram of soil. These density levels were chosen based on general thresholds for PCN management actions.
For the AMF inoculum, commercially available Rhizophagus irregularis inoculum was added to each pot. The control treatment received a blank inoculum to account for any nutrients present. Each treatment had six biological replicates.
One week after planting, plant height and chlorophyll content were measured at weekly intervals. Leaves of similar size from each plant's canopy were sampled. After 12 weeks of growth, plants were harvested, separated into shoots, roots, and tubers, and weight measurements were taken for each plant component. These samples were then freeze-dried for approximately three days.
Roots were cleaned and sub-samples were taken for quantification of AM root length colonization and stored for further use.
To estimate the number of nematode cysts, the pot soil was mixed, and cysts were extracted using Fenwick's (1940) method. Unhatched second-stage juveniles were counted to determine the number of eggs per cyst, which reflects the reproductive capacity of the nematodes under each treatment.
Plant roots were stained using the 'ink and vinegar' staining method. Root samples were processed, and AM fungal structures were stained with ink and vinegar. The percentage of root length colonized by AMF was assessed.
AM fungal hyphae were extracted from bulk soil, stained with ink and vinegar, and their lengths per pot were calculated.
A random subsample of potato roots collected from the field was processed for fungal barcoding via Next-Generation Sequencing of the Internal Transcribed Spacer II. The taxonomic classification of fungi within the root samples was provided by the sequencing provider, along with diversity indices.
The plant's height experienced a decrease when exposed to 125 G. pallida eggs per gram of soil but remained unaffected at lower densities. AMF-colonized potatoes displayed increased shoot height compared to non-AMF plants at the same G. pallida density over the growth period. The timing of when AMF plants surpassed the height of G. pallida-only plants depended on the G. pallida density, with higher G. pallida densities delaying the AMF-induced growth increase. The shoot height at 8 weeks showed a positive correlation with tuber yields.
Chlorophyll content, a crucial parameter for plant productivity and stress indicators, was measured throughout plant development. Generally, chlorophyll content decreased as plants aged. However, AMF-inoculated plants at G. pallida densities of 35 or lower displayed increased chlorophyll content in their leaves compared to non-AMF plants. This difference was particularly noticeable around week 6.
AMF colonization also led to an increase in the number of eggs per cyst at initial G. pallida densities of 35 eggs or lower. However, at a G. pallida density of 125 eggs per gram of soil, more nematodes developed into cysts, but they had fewer eggs per cyst compared to lower density treatments, and the presence of AMF did not significantly impact this outcome.
Notably, the presence of G. pallida at lower densities increased AMF colonization of roots compared to treatments without G. pallida. Conversely, a G. pallida density of 125 eggs per gram of soil resulted in a greater amount of AMF hyphae in the soil compared to controls without G. pallida.
The extent of mycorrhizal (AMF) colonization in the potato crop was evaluated upon harvest to assess the effectiveness of AMF inoculation in field soil. Both inoculated and non-inoculated plots exhibited AMF colonization of potato roots. However, the inoculated plots showed a higher degree of root colonization by AMF. The application of the nematicide nemathorin and subsequent reduction in G. pallida densities did not affect AMF colonization of roots in the field.
An analysis of fungal barcoding from root tissue samples before harvest revealed that the addition of AMF inoculum did not impact the total number of fungal operational taxonomic units (OTU) associated with the root. Still, it did increase their diversity (Shannon index). Notably, the AMF detected in non-inoculated plots was identified as Funneliformis sp., suggesting that this genus is native to the field. Overall, there was a higher relative abundance of Glomeromycetes in samples that had received the AMF inocula.
While Clarioeoglomus sp., Glomus sp., and Diversispora sp. were present in the commercial inoculum applied to the soil, they were not identified as associated with potato roots. Only Rhizophagus irregularis and Funneliformis sp. were detected in the roots. The inclusion of Funneliformis sp. within the inoculum did not increase its relative abundance in the roots compared to non-inoculated plots containing native soil fungi. Fungal barcoding indicated that the increased AMF content in inoculated roots was due to the presence of R. irregularis.
G. pallida cyst and egg counts were lower in plots treated with nematicide compared to non-nematicide plots both before planting and after harvest. AMF inoculation did not impact cyst counts but did enhance G. pallida fitness in terms of egg production. This increase in G. pallida egg content was more pronounced in soils treated with nematicide (lower G. pallida densities) compared to non-nematicide controls (higher G. pallida densities).
At harvest, plots treated with nematicide produced a greater yield, on average, with a 16% increase compared to non-nematicide plots. While AMF inoculation did not affect the total yield tonnage, it led to larger harvested tubers compared to non-inoculated plots.
The multiplication rate of G. pallida is heavily influenced by the resistance of the cultivated potato variety, leading to long-term implications due to the nematode's persistence in agricultural soils, even in the absence of a host. Factors like soil temperature, rainfall, and the cultivation of susceptible or resistant/non-host plants can impact nematode density in fields. Higher densities of G. pallida result in greater yield losses and limit growers' variety choices, forcing them to select options that can tolerate higher nematode burdens.
In fields infected by G. pallida, potato roots are often simultaneously colonized by various soil-borne symbionts, including arbuscular mycorrhizal fungi (AMF). AMF are widespread in soils and form mutualistic relationships with plants, aiding in nutrient uptake. Recent studies have shown that when potato roots are co-colonized by G. pallida and AMF, there is a shift in carbon allocation, with AMF partners receiving less while nutrient transfer to the host remains stable. This presence of AMF in co-colonized plants seems responsible for boosting G. pallida fitness and enhancing host plant tolerance to the nematode.
However, these findings were based on a single density of G. pallida on plant roots. In the field, G. pallida infection densities can vary, influencing resource allocation. High G. pallida densities can significantly impact host plant tolerance. The impact of G. pallida density on AMF-induced tolerance in potatoes remains uncertain. While AMF appear to increase G. pallida populations and improve plant vigor by enhancing nutrient supply, this may not hold under high G. pallida densities, potentially leading to crop failure and persistently high G. pallida populations.
Furthermore, AMF colonization of the host can also increase populations of other plant-parasitic nematodes, expanding the problem. Field trials are exploring the use of AMF as soil amendments to improve yields, enhance the biosynthesis of desirable phytochemicals, and increase plant tolerance to pests and abiotic stresses. This approach offers an alternative to heavy pesticide use, which faces restrictions for environmental reasons. Field trials have shown promising results, particularly in potato cultivation. However, the effectiveness of AMF applications can vary depending on factors like plant and fungal genotypes, compatibility issues, competition with native species, field capacity for AMF, and farming practices.
To date, only a limited number of field trials have investigated the application of AMF inoculum to enhance plant tolerance to plant-parasitic nematodes. Some experiments have shown AMF to reduce infection and enhance host plant tolerance to root-knot nematodes. Field trials with cyst nematodes suggest that AMF can promote hatching and potentially work synergistically with nematicides to reduce nematode populations in the soil. Whether AMF can effectively mitigate the effects of cyst nematode pests in field environments remains a topic requiring further investigation.
In this work, greenhouse and field experiments were conducted to assess the impact of G. pallida density on AMF-induced tolerance and G. pallida fitness. Additionally, we examined the influence of a commercial AMF inoculant on the native AMF community.
Methods
Greenhouse methods
Potato tubers were planted in pots with a mixture of sterilized sand and topsoil (50:50 ratio). Various treatments were applied, including different densities of G. pallida and the addition of AMF inoculum. G. pallida-free controls were also included.All plants were grown in a controlled environment in a containment glasshouse with specific conditions (18–20°C) temperature and 16-hour day length) and received regular watering. G. pallida inoculum was created by infecting plants with Globodera pallida cysts and grown for 12 weeks with regular fertilizer applications until plant death to increase G. pallida populations for later use.
The PCN content of the inoculum soils was determined through Fenwick's (1940) method and mixed with sterilized sand and loam topsoil to achieve desired PCN densities of 15, 35, and 125 eggs per gram of soil. These density levels were chosen based on general thresholds for PCN management actions.
For the AMF inoculum, commercially available Rhizophagus irregularis inoculum was added to each pot. The control treatment received a blank inoculum to account for any nutrients present. Each treatment had six biological replicates.
One week after planting, plant height and chlorophyll content were measured at weekly intervals. Leaves of similar size from each plant's canopy were sampled. After 12 weeks of growth, plants were harvested, separated into shoots, roots, and tubers, and weight measurements were taken for each plant component. These samples were then freeze-dried for approximately three days.
Roots were cleaned and sub-samples were taken for quantification of AM root length colonization and stored for further use.
To estimate the number of nematode cysts, the pot soil was mixed, and cysts were extracted using Fenwick's (1940) method. Unhatched second-stage juveniles were counted to determine the number of eggs per cyst, which reflects the reproductive capacity of the nematodes under each treatment.
Plant roots were stained using the 'ink and vinegar' staining method. Root samples were processed, and AM fungal structures were stained with ink and vinegar. The percentage of root length colonized by AMF was assessed.
AM fungal hyphae were extracted from bulk soil, stained with ink and vinegar, and their lengths per pot were calculated.
Open field method
To validate the glasshouse data in agroecosystems, a field trial was established. The trial location was in Bensgate, Spalding, UK. Potatoes were planted with inoculum containing various fungal species. Some plots received nematicide treatment to reduce G. pallida populations, while others served as fallow plots. Data was collected and samples were harvested following standard procedures.A random subsample of potato roots collected from the field was processed for fungal barcoding via Next-Generation Sequencing of the Internal Transcribed Spacer II. The taxonomic classification of fungi within the root samples was provided by the sequencing provider, along with diversity indices.
Results
Greenhouse results
Influence of G. pallida population density on host growth and AMF-induced host resilience
In the glasshouse trial, the potato plant's tuber yield was assessed to understand the influence of varying G. pallida densities in the presence of AMF. Notably, greater tuber yields were observed in plants with AMF, without G. pallida infection. However, G. pallida infection led to a reduction in yields in a density-dependent manner, with higher G. pallida densities resulting in diminished potato yields. AMF colonization proved beneficial for yields, even when G. pallida densities were below 15 eggs per gram of soil. A similar pattern was observed for shoot biomass, where mycorrhizal inoculation enhanced shoot biomass at all densities except when G. pallida density was at 35 G. pallida eggs per gram of soil.The plant's height experienced a decrease when exposed to 125 G. pallida eggs per gram of soil but remained unaffected at lower densities. AMF-colonized potatoes displayed increased shoot height compared to non-AMF plants at the same G. pallida density over the growth period. The timing of when AMF plants surpassed the height of G. pallida-only plants depended on the G. pallida density, with higher G. pallida densities delaying the AMF-induced growth increase. The shoot height at 8 weeks showed a positive correlation with tuber yields.
Chlorophyll content, a crucial parameter for plant productivity and stress indicators, was measured throughout plant development. Generally, chlorophyll content decreased as plants aged. However, AMF-inoculated plants at G. pallida densities of 35 or lower displayed increased chlorophyll content in their leaves compared to non-AMF plants. This difference was particularly noticeable around week 6.
Reduced influence of AMF-triggered G. pallida health as G. pallida density rises
The study also examined the effect of different initial G. pallida densities on the previously reported increase in G. pallida infection and reproduction induced by AMF. AMF colonization increased the number of G. pallida cysts recovered at harvest, particularly in plants with initial G. pallida densities of 35 eggs or lower. The increase in cysts was more pronounced at lower initial G. pallida densities.AMF colonization also led to an increase in the number of eggs per cyst at initial G. pallida densities of 35 eggs or lower. However, at a G. pallida density of 125 eggs per gram of soil, more nematodes developed into cysts, but they had fewer eggs per cyst compared to lower density treatments, and the presence of AMF did not significantly impact this outcome.
Notably, the presence of G. pallida at lower densities increased AMF colonization of roots compared to treatments without G. pallida. Conversely, a G. pallida density of 125 eggs per gram of soil resulted in a greater amount of AMF hyphae in the soil compared to controls without G. pallida.
Open field results
Impact of AMF Colonization on Potato Growth and PCN Fitness in the Field
A field trial was conducted to validate the findings observed in the glasshouse within real agro-ecosystems. Despite generally dry conditions during the season, increased rainfall and temperatures shortly after planting in April provided favorable conditions for the potato crop.The extent of mycorrhizal (AMF) colonization in the potato crop was evaluated upon harvest to assess the effectiveness of AMF inoculation in field soil. Both inoculated and non-inoculated plots exhibited AMF colonization of potato roots. However, the inoculated plots showed a higher degree of root colonization by AMF. The application of the nematicide nemathorin and subsequent reduction in G. pallida densities did not affect AMF colonization of roots in the field.
An analysis of fungal barcoding from root tissue samples before harvest revealed that the addition of AMF inoculum did not impact the total number of fungal operational taxonomic units (OTU) associated with the root. Still, it did increase their diversity (Shannon index). Notably, the AMF detected in non-inoculated plots was identified as Funneliformis sp., suggesting that this genus is native to the field. Overall, there was a higher relative abundance of Glomeromycetes in samples that had received the AMF inocula.
While Clarioeoglomus sp., Glomus sp., and Diversispora sp. were present in the commercial inoculum applied to the soil, they were not identified as associated with potato roots. Only Rhizophagus irregularis and Funneliformis sp. were detected in the roots. The inclusion of Funneliformis sp. within the inoculum did not increase its relative abundance in the roots compared to non-inoculated plots containing native soil fungi. Fungal barcoding indicated that the increased AMF content in inoculated roots was due to the presence of R. irregularis.
G. pallida cyst and egg counts were lower in plots treated with nematicide compared to non-nematicide plots both before planting and after harvest. AMF inoculation did not impact cyst counts but did enhance G. pallida fitness in terms of egg production. This increase in G. pallida egg content was more pronounced in soils treated with nematicide (lower G. pallida densities) compared to non-nematicide controls (higher G. pallida densities).
At harvest, plots treated with nematicide produced a greater yield, on average, with a 16% increase compared to non-nematicide plots. While AMF inoculation did not affect the total yield tonnage, it led to larger harvested tubers compared to non-inoculated plots.
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