The beneficial root endophyte Piriformospora indica reduces egg density of the soybean cyst nematode

Biological Control 90 (2015) 193–199

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The beneficial root endophyte Piriformospora indica reduces egg density of the soybean cyst nematode Ruchika Bajaj a,b, Weiming Hu c, YinYin Huang b, Senyu Chen c,d, Ram Prasad a, Ajit Varma a, Kathryn E. Bushley b,⇑ a

Amity Institute of Microbial Technology, Amity University Uttar Pradesh, Sector 125, Noida 201303, India Department of Plant Biology, University of Minnesota, Saint Paul, MN, 55108, United States Institute of Microbiology, Chinese Academy of Sciences, Beijing, China d Southern Research and Outreach Center, University of Minnesota, Waseca, MN 56093, United States b c

h i g h l i g h t s

g r a p h i c a l a b s t r a c t

 Soil was inoculated with root

endophyte Piriformospora indica.  A significant decrease in egg

population density observed in P. indica amended soil.  P. indica is a promising biocontrol agent of the SCN.

a r t i c l e

i n f o

Article history: Received 20 February 2015 Revised 29 May 2015 Accepted 31 May 2015 Available online 2 July 2015 Keywords: Piriformospora indica Soybean Soybean cyst nematode Biotic stress Biocontrol

⇑ Corresponding author. E-mail address: [email protected] (K.E. Bushley). http://dx.doi.org/10.1016/j.biocontrol.2015.05.021 1049-9644/Ó 2015 Elsevier Inc. All rights reserved.

a b s t r a c t The soybean cyst nematode (Heterodera glycines) is a plant parasitic nematode that is a major plant pest worldwide and causes severe economic and yield losses. Piriformospora indica, a plant growth promoting fungus isolated from the Thar Deserts of western India, has been shown to protect a wide range of plants from various biotic and abiotic stresses. To evaluate the potential of P. indica to protect soybean (Glycine max) seedlings from damage by the soybean cyst nematode (SCN), we amended soil with two different concentrations of P. indica (2.5% and 5% w/w) and inoculated with second-stage juveniles (J2s) of SCN in each treatment. After 60 days, abundance of nematode eggs was measured by calculating SCN egg population densities. We found that egg density/100 cc soil was significantly decreased by 29.7% and 36.7% respectively in the soil amended with 2.5% and 5% P. indica compared to a control. Amendment with P. indica also had a strong growth and yield promoting effect in Soybean. Although root biomass was significantly decreased by 27.9% and 33.5% in the two treatments compared to the control, shoot biomass (dry weight) increased by 30.8% and 8.2% in the 2.5% and 5% P. indica treatments compared to the control. Additionally, plant development was accelerated and a 75% increase in flowering was observed between the 2.5% P. indica treatment and the control. We conclude that P. indica used as a soil amendment decreases abundance of the SCN in soil and has plant-growth promoting properties that may help offset yield losses due to plant parasitic nematodes. Ó 2015 Elsevier Inc. All rights reserved.

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1. Introduction The soybean cyst nematode (SCN), Heterodera glycines, is a plant-parasitic nematode and a destructive pest of soybean worldwide (Monson and Schmitt, 2004). The life cycle of the SCN has three stages: egg, juvenile, and adult. Soybeans are infected by the second-stage juveniles (J2s), the microscopic colorless worm stage that penetrates the roots with a stylet. After invading the roots, the nematodes migrate towards the vascular tissue where they feed and develop. Feeding causes changes in internal root structure and interferes with normal root function, causing plant disease (Lambert and Bekal, 2002). Approximately three weeks after infection, under optimum conditions (soil temperatures at 27–29 °C), female juveniles that are fertilized by males grow into mature females. The enlarged females burst through the root surface and lay eggs in a jelly like mass attached to their posterior end, retaining about two-thirds of the eggs within their swollen bodies. After the female dies, the cuticle becomes melanized to form a brown cyst that encloses approximately 200–400 eggs. The cysts protect the eggs from desiccation, chemicals, predators, and some parasites (Duan et al., 2009). The SCN is able to tolerate various environmental stresses, especially low temperature, primarily due to protection of eggs within the cysts. The SCN causes the greatest yield loss worldwide of any disease or pest of soybean, with losses in the United States alone estimated at 1 billion US dollars annually (Riggs, 2004; Arelli and Wang, 2008). Various strategies have been employed to suppress the damage caused by the SCN (Porter et al., 2001). These include crop rotation and other cultural practices, resistant cultivars, and nematicides (Koenning et al., 1993; Riggs and Schuster, 1998; Schmitt, 1991; Young and Hartwig, 1992; Young, 1998a,b). Ross (1962) reported that crop rotation with non-hosts of the SCN such as corn (Zea mays), wheat (Triticum aestivum), or grain sorghum (Sorghum bicolor) was an effective management strategy. However, resistant cultivars are costly to produce and the SCN may evolve resistance within a short period of time (Zheng and Chen, 2011). While a number of highly effective chemical nematicides, including fumigants and non-fumigants, have been deployed over the past 30 years, the most effective compounds (e.g., methyl bromide) have been banned or restricted due to environmental and health concerns, as many are toxic to mammals, including humans. Various fungal and bacterial pathogens of nematodes have also been employed as potential biocontrol agents, with variable or limited success (Chen and Dickson, 2012). Consequently, plant-parasitic nematodes are currently among the most difficult crop pathogens to manage and there is a great need for development of nontoxic, inexpensive, and effective control methods. Piriformospora indica is a root endophyte that was isolated from the Thar Desert of western India (Verma et al., 1998; Varma et al., 1999). This fungus has growth promoting effects mimicking those of arbuscular mycorrhizal fungi (Mishra et al., 2014; Chadha et al., 2014) and increases biomass and yield of many plant species (Malla et al., 2004; Varma et al., 2014). It colonizes a wide range of plants including gymnosperms, angiosperms and orchids (Ye et al., 2014) and improves growth through increased nutrient uptake (N and P) of the host plants (Sherameti et al., 2005; Yadav et al., 2010). It has also been shown to enhance the production of protective secondary metabolites like podophyllotoxins in Linum album (Baldi et al., 2008), bacosides in Bacopa monniera (Prasad et al., 2008a, 2013), and curcumin and volatile oils in Curcuma longa (Bajaj et al., 2014). All these compounds may induce local and systemic resistance (Deshmukh et al., 2006), providing increased resistance to biotic stresses such as cyst nematodes and other plant pathogens as well as abiotic stresses such as acidity, heavy metals and drought (Vadassery et al., 2009a,b). The

fungus has been shown to confer resistance against root and leaf fungal pathogens including Fusarium culmorum and Blumeria graminis in barley by increasing antioxidant activity (Waller et al., 2005). It also decreased disease symptoms of F. culmorum, Pseudocercosporella herpotrichoides, and B. graminis on wheat (Deshmukh and Kogel, 2007; Serfling et al., 2007). Recently, Daneshkhah et al. (2013) reported that inoculation of P. indica onto Arabidopsis roots in vitro antagonized the infection and development of cyst nematodes. In this study, we tested the ability of P. indica amended to soil of soybean plants to decrease reproduction, as measured by egg density, of the SCN. 2. Materials and methods 2.1. Fungus cultivation P. indica ATCC (204458) was cultured in potato dextrose broth with constant shaking at 100 rpm at 30 °C. The mycelium was harvested 8 days after inoculation by filtration to remove liquid media. 2.2. Preparation of soil Field soil was collected from an agricultural field with no soybean cyst nematode infestation at the Southern Outreach and Research Station in Waseca, Minnesota, USA. Soil was mixed with 30% sand and autoclaved at 121 °C for 60 min. Mycelium of P. indica at concentrations of 2.5% (w/w) and 5% (w/w) was thoroughly mixed with soil and placed into four 16-cm-diameter clay pots. A control treatment consisting of the autoclaved soil mixture with no P. indica amendment was similarly prepared. Eight soybean seeds were surface sterilized with 0.5% NaOCl for 3 min, rinsed three times in sterile water, and sown in each pot. One week after planting, seedlings were thinned to keep only five seedlings of approximately the same size and developmental stage per pot. The pots were arranged randomly and maintained in a greenhouse with a temperature ranging from 26 °C ± 4 °C with 16 h light/8 h dark. Pots and seedlings were watered daily. 2.3. Preparation and inoculation of J2s SCN race 3 nematodes, which were originally collected from a field at the Southern Outreach and Research Station in Waseca, Minnesota, USA, were cultured on soybean plants in sterilized soil in a greenhouse. A soil suspension containing newly formed cysts was poured into a 2-liter jug and sprayed with a strong jet of water. This suspension was allowed to settle for 5–10 s and then poured onto a 850-lm-pore (#20) sieve nested on top of a 250-lm-aperture (#60) sieve. This procedure was repeated 5 times to ensure that all cysts were collected. The cysts were then sprayed with water on the 850-lm-aperture screen to remove root debris and collected onto a 250-lm-aperture sieve. The cysts were washed from the 250-lm-aperture sieve into a 50 mL centrifuge tube with 63% (w/v) sucrose and centrifuged at 1100g for 5 min. The cysts floating on the top of these tubes were collected into a tube mounted with a 150-lm-pore screen, and eggs were released by crushing the cysts on a 150-lm-aperture sieve with a rubber stopper mounted on a motor (Faghihi and Ferris, 2000). The eggs were cleaned and separated from debris by centrifugation in a 38% (w/v) sucrose solution for 5 min at 1500g to remove most of the remaining debris. The collected eggs were transferred onto a 38-lm-aperture sieve, rinsed with sterile water, treated with SCQ antibiotic solution (100 ppm streptomycin sulfate, 50 ppm chlorotetracycline, and 20 ppm 8-quinolinol) for 24 h at 4 °C. They were then placed on a 35-lm-aperture nylon cloth on a screen immersed in 4 mM ZnCl2 solution to hatch J2s. The

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temperature of the containers was maintained at approximately 22–24 °C. The viability and concentration of J2s was determined under an inverted microscope. Suspensions of J2s were then inoculated into approximately 5 cm deep holes dug adjacent to the root systems of each seedling with a 1 mL pipet tip. Approximately 600 juveniles per plant for a total of 3000 juveniles per pot were inoculated 1 week after planting. 2.4. Plant growth measurements Plants were harvested 60 days after inoculation of juveniles. The plant height was measured in metric scale using a measuring tape alongside each plant. Observations were recorded in four independent replicate pots, each containing five plants. The mean of the five plants per replicate pot was calculated and used for statistical comparisons. The shoots of soybean were harvested and washed with water. The roots were washed under running tap water to remove the adhered soil. Fresh weight was recorded at harvest. Shoots and roots were then dried in a plant drier and dry weight recorded. 2.5. Chlorophyll content Chlorophyll meter readings (SPAD values) were taken at the center of three leaves from each plant with a Konica-Minolta SPAD-502 chlorophyll meter 60 days after planting. The mean of three leaves per plant was used to calculate the average chlorophyll content for each plant. The mean chlorophyll content of all five plants in each replicate pot was then used for statistical comparisons between treatments. 2.6. Root colonization Roots were washed in running tap water, cleaned by soaking in 10% KOH for 4 days, acidified with 1 N HCl for 5 min, and then stained with lacto-phenol cotton blue. The roots were observed under 630X optical microscope (Olympus BH2, Leeds Precision Instrument, Inc.). Estimation of percent colonization was done using the grid line-intersect method (Norris et al., 1994; Varma and Kharkwal, 2009). Spores were counted under a stereo binocular microscope. Percentage root colonization was calculated with the formula: percent colonization = number of colonized roots  100/number of roots observed. 2.7. Measurement of egg population density Plants were harvested 60 days after inoculation of juveniles. The soil was thoroughly mixed, and a subsample of 100 cc soil (weight of approximately 190 g of soil  100 cc soil) was collected in a 1-liter beaker and soaked in 500 ml water for 30 min. The eggs were isolated and cleaned using the procedure described above. The eggs were then suspended in 50-mL water and the number of nematode eggs in a 200 lL subsample was counted using an inverted microscope in a petri-plate marked with a grid. The total number of eggs per 100 cc soil was calculated with the following formula: total number of eggs = number of eggs counted in sample volume  total volume of egg suspension/sample volume of egg suspension counted. 2.8. Statistical analyses To assess statistical significance of differences between treatments, each P. indica treatment (2.5% and 5%, respectively) was compared independently to the control using a two-sided students t-test (Sokal and Rohlf, 1995) implemented in R (R Core Team, 2013). The module t.test, which automatically adjusts for any

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unequal variance through use of the Welch approximation for degrees of freedom, was used for all comparisons. The mean value of all five plants for each of the four replicate pots per treatment was used as input to the t-test, and a p-value of .05 or less was considered statistically significant. 3. Results 3.1. Colonization of soybean roots by P. indica In this study, P. indica was inoculated onto the oilseed crop soybean in a controlled greenhouse trial in order to analyze its possible effects on growth, development, and pest resistance towards the SCN. Staining of soybean roots with lactophenol cotton blue revealed extensive root colonization by P. indica and formation of intracellular chlamydospores (Fig. 1B). However, colonization was confined to the root cortex. Chlamydospores were found as isolated spores, pairs, tetrads, long chains, and sometimes in clusters. Levels of root colonization ranged from 45% to 50% at 60 days after planting. 3.2. Effects of P. indica on growth and development of soybean Soybean showed a positive interaction with P. indica, as demonstrated by increased shoot biomass and length of inoculated plants as compared to control plants (Figs. 1E, 2, Table 1). Shoot length increased by 17.4% in the 2.5% P. indica treatment compared to the control (Fig. 2, Table 1). Similarly, the average shoot dry weight increased by 30.8% in the 2.5% P. indica treatment compared to control and by 8.2% in the 5% P. indica treatment (Fig. 2, Table 1). In contrast, the overall weight of colonized roots was lower than the weight of the uncolonized control roots. The root dry weights in the 2.5% and 5% P. indica treatments decreased by 27.9% and 33.5%, respectively, as compared to the control (Figs. 1C and D, 2, Table 1). There was no detectable effect of the fungus on the chlorophyll content (Fig. 2, Table 1). Inoculation with P. indica also affected timing and extent of plant development. The average number of leaves and branches increased by 17.2% and 14.9%, respectively, in the 2.5% treatment as compared to control, although these differences were not statistically significant (Fig. 3, Table 2). We also observed that P. indica not only induced development of the vegetative aerial part of the plant, but was also responsible for early maturation with respect to flowering. There was an increase of more than 75% in flowers observed in the 2.5% treatment compared to the control (Fig. 3, Table 2). Similar results were observed for the 5% P. indica treatment with an increase of 13.5%, 10.6% and 24% in number of leaves, branches and flowers respectively (Fig. 3, Table 2). 3.3. Effect of P. indica on SCN egg density The number of SNC eggs per cc soil, a common screening measure of SCN severity in agricultural fields, was significantly lower in the P. indica amended pots (Fig. 4A, Table 3). There was a decrease of 29.7% in the 2.5% P. indica treatment and 36.7% in the 5% P. indica treatment (Fig. 4A, Table 3). Egg density per cc soil was also significantly reduced by 11.0% between the 2.5% to the 5% P. indica treatment (Fig. 4A, Table 3). Egg density calculated as number of eggs/cc soil/gram root wet or dry weight also showed a trend of decreasing egg density with increasing P. indica in soil, although these comparisons were not statistically significant. For egg density/cc soil/gram root wet weight, the egg density decreased by 3.5% from control to 2.5% P. indica and by 7.1% from 2.5% to 5% P. indica. For egg density/cc soil/gram root dry weight, these decreases were 3.9% and 1.9%, respectively (Fig. 4B, Table 3).

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Fig. 1. (A) Staining of control roots, (B) staining of P. indica colonized roots showing chlamydospores (arrows) growing within soybean roots. Roots were cleared first for 4 days in KOH and stained with lactophenol blue for 15 min, (C) root system of control plants showing greater biomass and extensive branching, (D) root system of plant from 5% treatment showing decreased biomass and branching, (E) shoots of control (left) and 2.5% P. indica treatment (right) showing increased shoot length, biomass, and flowering.

Fig. 2. Plant growth and chlorophyll content. While chlorophyll content did not change significantly across treatments, shoot length and shoot dry weight increased significantly in the 2.5% treatment. Shoot dry weight but not shoot length increased significantly in the 5% treatment. Consistent with previous studies, root weight decreased significantly in both P. indica treatments compared to the control. An asterisk above indicates a significant (p < .05) increase or decrease compared to the control.

4. Discussion P. indica colonizes a wide range of plants and can be cultivated easily on various artificial media and within roots, forming pear-shaped chlamydospores (Varma et al., 2012; Chadha et al., 2014). In this study, the fungus colonized only the cortex of soybean roots in a greenhouse study. Similarly, in Oryza sativa L., cytological studies of colonized roots revealed that the fungal mycelium penetrates into the epidermis and cortical cells but never reaches the endodermis layer and stelar tissue (Bagheri et al., 2013). The soybean cyst nematode is a destructive plant pest worldwide. As a soil-borne disease for which chemical controls are highly toxic, it is very difficult to control (Duan et al., 2009).

Development of resistant soybean cultivars is currently the most effective management strategy to control the SCN but has obvious shortcomings, such as the long time and significant resources required to breed resistant cultivars compared to the relative ease with which resistance can be lost (Yu, 1998). Previous studies of inoculation of Arabidopsis thaliana roots with P. indica in an in vitro petri plate assay, provide support for direct inhibition of nematodes by P. indica. Fungal culture filtrate, as well as application of P. indica cell wall extract, significantly decreased the motility, infectivity, development and reproduction of the beet cyst nematode (Heterodera schachtii) (Daneshkhah et al., 2013). In this study, we investigated the potential of this fungus to protect soybean from damaging effects of the SCN in a more realistic greenhouse pot study. Our results demonstrate that inoculation

R. Bajaj et al. / Biological Control 90 (2015) 193–199 Table 1 Indicators of plant growth and nutrient status. Growth parameter

Control

2.5% P. indica Com

5% P. indica

% Root colonization

0.00 n/a

46.04 ±0.03

48.00 ±0.02

Shoot Length (cm)

38.16 ±3.40

44.80⁄ ±2.78

38.86 ±1.39

Chlorophyll (SPAD)

28.78 ±0.42

29.38 ±0.63

28.80 ±0.54

Root fresh weight (gm)

37.03 ±4.03

26.78⁄ ±1.03

25.87⁄⁄ ±2.24

Root dry weight (gm)

11.97 ±1.52

8.63⁄ ±0.69

7.96⁄⁄ ±1.01

Shoot fresh weight (gm)

43.91 ±4.14

55.13 ±8.01

47.50 ±2.71

Shoot dry weight (gm)

13.73 ±1.00

17.97⁄⁄ ±0.99

16.11⁄ ±0.95

Mean (top) and one standard deviation (bottom) for each parameter. An * indicates a statistically significant difference between that treatment compared to the control at p = .05 and an ** indicates significance at p = .01.

with P. indica had a strong growth promoting effect in soybean, significantly increasing aboveground biomass, shoot:root ratio, and flowering (Figs. 2 and 3, Tables 1 and 2). We also observed a significant decrease in egg density/cc soil in both the 2.5% and 5% P. indica treatments (Fig. 4A, Table 3). However, root weight and volume also decreased significantly in P. indica treated plants (Fig. 2, Table 1). While egg density calculations normalized by root weight (egg density/cc soil/gram root weight) also showed a trend of decreasing egg density with higher levels of P. indica in soil, these comparisons were not statistically significant (Fig. 4B, Table 3). Here we demonstrate that soybean plants colonized with P. indica were able to sustain higher biomass production and flowering while simultaneously decreasing SCN egg densities in bulk soil, thus providing a promising approach for management of the SCN. Although a precise mechanism of inhibition remains unknown, P. indica may either directly inhibit nematode development or may control plant responses that impact nematode colonization and development. The fungus has been shown to control expression of a Nicotiana attenuate homolog of HslPro-l, a locus initially

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identified as having a role in resistance to the beet cyst nematode (H. schachtii Schmidt) (Cai et al., 1997), but now thought to be involved in more generalized responses to both abiotic and biotic stresses and potentially in repartitioning of carbon resources within the plant (Schuck et al., 2012). The role of sucrose synthases and invertases may also impact the development of cyst nematodes through changes in syncytial sugar levels that alter sink strength and thus change systemic sugar partitioning. Elevations in sugar levels were shown to contribute substantially to enhanced nematode development (Cabello et al., 2013) and have major nutritional value for this obligate parasite. Thus, allocation to shoot growth over root growth, as observed in this study, could impact the availability of nutritional carbon sources available to root cyst nematodes. Alternatively, decreased root biomass and branching observed in P. indica colonized plants (Figs. 1 and 2, Table 1) may decrease the root surface area available for colonization by the SCN and thus impact both numbers of infections and cyst and egg formation. The fungus has previously been shown to decrease root volume through apoptotic cell-death in root cells during fungal colonization (Deshmukh et al., 2006). However, decreased root growth does not negatively impact plant growth and instead colonization with P. indica has been shown to increase plant growth in a number of other economically important crops, including Barley (Zuccaro et al., 2009), wheat (Serfling et al., 2007), rice (Das et al., 2014), maize (Kumar et al., 2009), and sunflower (Bagde et al., 2011). Plant biomass increases as well as stimulation of secondary metabolites has also been observed in a number of medicinal plants such as B. monniera (Sahay and Varma, 1999; Prasad et al., 2013), Withaniasomnifera, Spilanthescalva, Chlorophytum borivilianum (Rai et al., 2001; Prasad et al., 2008b), Adhatodavasica (Rai and Varma, 2005), and C. longa (Bajaj et al., 2014). Significant increases in both shoot length and biomass with P. indica were observed in this study (Figs. 1 and 2, Table 1). The increased growth of P. indica-colonized plants could be associated with enhanced nutrient uptake (especially of phosphorus and nitrogen) from the soil, similar to the nutrient enhancement by AM fungi (Smith and Smith, 1997). It has been shown that P. indica is involved in the transportation of phosphate to the host plant through a specialized phosphate transporter (Yadav et al., 2010). Several studies have demonstrated that

Fig. 3. Indicators of plant development. Numbers of branches and leaves increased in P. indica treatments, although not significantly. The number of flowers were significantly greater in both the 2.5% and 5% P. indica treatments. An asterisk above indicates a significant (p < .05) increase or decrease compared to the control.

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Table 2 Indicators of plant development. Development parameter

Control

2.5% P. indica

5% P. indica

No branches

11.8 ±1.5

13.5 ±1.2

13.0 ±0.8

No. leaves

35 ±4.5

41 ±5.5

40 ±4.6

No. flowers

22.5 ±2.6

39.5⁄⁄ ±2.5

28⁄⁄ ±1.4

Mean (top) and one standard deviation (bottom) for each parameter. An ** indicates a statistically significant difference for that treatment compared to the control at p = .01.

been shown to shorten the time to flowering and increase the number of flowers and seeds (Barazani et al., 2005; Rai et al., 2001; Shahollari et al., 2007), consistent with the higher numbers of inflorescences that were observed in the P. indica colonized soybean plants in this study (Fig. 3, Table 2). Thus, colonization of soybean by P. indica stimulated growth and was able to offset any biomass and yield decreases due to the SCN. 5. Conclusion Of the microorganisms that parasitize or prey on nematodes, fungi hold an important position and some of them have shown great potential as biocontrol agents (Jatala, 1986; Stirling, 1991). Amendment of soil with P. indica appeared to reduce egg production by the SCN while also stimulating growth and development of soybean plants. While further research is needed to address the specific mechanisms by which P. indica is able to both lower levels of SCN eggs in soil while also enabling soybean plants to sustain higher yields in the presence of the SCN, it appeared that the fungus has potential applications for controlling levels of SCN infestation in agricultural soils. Importantly, the generalized plant growth promoting properties of P. indica may help offset any yield losses due to nematodes and other plant pests. As the fungus is also non-pathogenic to other crops and higher animals and is easily cultivable in batch cultures at suitable pH and temperature ranges on artificial media and can be easily formulated for field application, it shows promise for the management of plant-parasitic nematodes such as the SCN. Acknowledgments The authors thank C. Johnson, and W. Gottschalk for technical assistance. Startup funds to Kathryn E. Bushley from University of Minnesota helped support this research. References

Fig. 4. (A) Number of nematode eggs found in 100 cc of soil decreased in both the 2.5% and 5% P. indica treatments compared to the control. The asterisk above a bar indicates a significant (p < .05) decrease. (B) Egg density calculated as eggs/gram of root wet weight also decreased in 2.5% and 5% P. indica treatments but these decreases were not significant.

colonization with P. indica also has strong effects on plant nitrogen metabolism, because the symbiotic interaction is accompanied by increased uptake of nitrate (Sherameti et al., 2005; Bajaj et al., 2014). In Arabidopsis roots infected with P. indica, gene expression was modified in the endoplasmic reticulum and plasma membrane and nitrate reduction in roots was stimulated (Peskan-Berghofer et al., 2004). The fungus also regulates the uptake and transport of important nutrients like Fe, Zn, Mg, and Cu. P. indica has also

Table 3 Effects of P. indica on SCN egg density. Egg density

Control

2.5% P. indica

5% P. indica

Eggs/100 cc soil

1.548  106 ±.105  106

1.088  106⁄⁄ ±.062  106

.980  106⁄⁄ ±.054  106

Eggs/gram root wet weight

.342  106 ±.045  106

.330  106 ±.028  106

.308  106 ±.019  106

Eggs/gram dry root weight

.106  106 ±.190  106

.102  106 ±.066  106

.100  106 ±.080  106

Mean (top) and one standard deviation (bottom) for each parameter. An * indicates a statistically significant difference for that treatment compared to the control at p = .05 and an ** indicates significance at p = .01.

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