Zaki A. Siddiqui* and Sumaiya Aziz
Received: September 08, 2020 Published: September 23,2020
Corresponding author: Zaki A. Siddiqui, Department of Botany, Aligarh Muslim University, India
Effects of silicon dioxide nanoparticles (SiO2 NPs) and Rhizobium ciceri was observed alone and in combination on Rhizoctonia
solani/ Fusarium solani / Meloidogyne incognita on the growth, photosynthetic pigments (chlorophyll and carotenoid) and proline
contents of Cicer arietinum.
Inoculation of M. incognita resulted in a greater reduction in plant growth, photosynthetic pigments and higher increase in proline contents than other test pathogens. Inoculation of R. ciceri or foliar spray of SiO2 NPs (0.10 mg ml-1) with pathogens under study resulted in increased plant growth, photosynthetic pigments and proline contents than without R. ciceri / NPs. Combined application of NPs with R. ciceri resulted in a greater increase in plant growth, photosynthetic pigments and proline contents in plants with pathogens than with NPs or R. ciceri. Plants without R. ciceri had a very poor root nodulation but nodulation was high in plants with R. ciceri. Pathogens under study and NPs had adverse effect on nodulation caused by R. ciceri. Wet rot and black root rot indices were 4 when R. ciceri and R. solani were inoculated respectively. R. ciceri / NPs reduced wet root rot, black root rot indices, galling and population of M. incognita. Disease indices, galling and population of M. incognita were reduced greatly by use of R. ciceri plus SiO2 NPs.
Keywords:Black Root Rot; Cicer Arietinum; Disease Index; Root Knot Nematode; Root Nodule Bacterium; Wet Root Rot
Chickpea (Cicer arietinum L.) is widely grown as major food
legume because of its rich nutrient values. It has high protein and
starch suitable for textile sizing Duke . Rhizobium Frank is root
nodule bacterium bacterium associated with roots of legumes for
symbiotic nitrogen fixation. Rhizobium inoculation has positive
effect on growth attributes, yield components and quality of
chickpea. Application of rhizobial isolates significantly suppress
root rotting fungi and root knot nematode Parveen et al.  and
reduced damaged caused Siddiqui and Mahmood .
Important diseases of chickpea include; Rhizoctonia wet rot (Rhizoctonia solani Kuhn) and Fusarium black root rot (Fusarium solani Mart. Amend Sacc.) and root-knot disease by Meloidogyne incognita (Kofoid and White) Chitwood. Parasitism by M. incognita in chickpea involves giant cells formation and root galling Vovlas et al. . Rhizoctonia wet root causes severe losses from seedling to maturity stages Jayalakshmi et al. . Its characteristic symptoms include root rotting and gradual yellowing and wilting of foliage, the rotted, discolored tissues are soft and wet. Fusarium black root rot also results in severe losses to of chickpea crop Jayalakshmi et al. . The root system is generally rotted with most of the finer roots destroyed, while the taproot remains intact, but dark and necrotic.
Silicon (Si) application is useful for increasing plant growth. Use of Si overcome biotic stresses and also improves plant’s physiological and mechanical properties Epstein ; Ma and Yamaji . Si also enhances disease resistance against pest and pathogens Marschner .
Frequent occurrence of F. solani, R. solani and M. incognita was observed in the chickpea fields of Aligarh, U.P. India. Infected plants were found with symptoms of diseases having poor plant growth. Silicon dioxide nanoparticles (SiO2 NPs) and Rhizobium ciceri were used alone and in combination management of F. solani, R. solani and M. incognita on chickpea. Effects of SiO2 NPs and R. ciceri were also observed on chickpea growth, photosynthetic pigments and also on proline contents.
Chickpea fields were surveyed for the collection disease materials of Aligarh U.P. Samples (root and soil) were collected and placed at 4oC until processing in refrigerator. The presence of plant parasitic nematodes and fungi in the collected samples were examined.
Roots showing disease symptoms were washed with sterilized water three times to remove soil particles. Isolation of fungi was made from infected plant parts Siddiqui et al.  on potato dextrose agar (PDA) medium. Identity of Rhizoctonia solani and Fusarium solani was confirmed. Later, pure cultures were separately prepared of both R. solani and F. solani
Root-knot nematodes females were dissected out from roots of chickpea having symptoms of root-knot. Later, perineal patterns were prepared for the identification of Meloidogyne sp. Taylor and sasser .
SiO2 NPs (Sigma-Aldrich, product No. 637246-50G) was used to prepare 0.10 mg ml-1 suspension by dissolving 100 mg nanapowder in 1-liter sterilized water. Foliar spray of 10 ml suspension was made on seedlings (15-day old) in a greenhouse with small spray pumps. Distilled water (10 ml) was sprayed on control plants and each treatment had five replications.
The effect of SiO2 NPs was studied on the M. incognita. Egg masses (20) of average size from eggplant roots were picked with sterilized forceps and effect of 0.10 mg ml-1 NPs on the hatching and mortality was observed as described by Siddiqui et al. .
Activity of SiO2 NPs against both fungi was observed separately. Ten ml NPs (0.10 mg ml-1 conc.) was added in 100 ml autoclaved PDA to observe effect as described by Siddiqui et al. 
Collected loam soil was sieved through 10 meshes. Clay pots were filled with 1 kg of soil mixed with loam soil and river sand (3:1). Before sterilization, soil surface of pots was wet with water, sterilized at 137.9 kPa for 20 minutes and allowed for cooling 24 hours.
Chickpea (Cicer arietinum L.) seeds Pusa-3022 were sterilized and sown in 15 cm pots Siddiqui et al. . Seedlings were inoculated and un-uninoculated control were placed in a glass house at 20 ± 2oC (Table 2). Pots were arranged on a bench and each treatment had five replications. Watering of plants was done as required. Ninety days after inoculation plants were harvested Inocula of fungi
Richard’s liquid medium was used for culturing of both fungi separately Riker and Riker  for obtaining inocula. Eighty ml liquid medium after filtration through muslin cloth was placed 250 ml flasks, autoclaved at 103.4 kPa for 15 minutes. Each fungus was separately inoculated, incubated, mycelia mats were collected, blotting sheets were used to remove excess water and inoculum of each fungus was prepared in Waring blender as described Siddiqui et al. . Ten ml of this suspension was used as inculum (1 g fungus).
Infected chickpea roots were used to collect Meloidogyne incognita, eggplants for its multiplication. Later, inoculum was prepared, juveniles were counted and 2000 juveniles were inoculated as described by Siddiqui et al. 
Rhizobium ciceri (100 g) charcoal culture was dissolved in 1 liter sterilized water. Ten ml (1 g inoculum) around the seeds per pot was inoculated where required.
Rhizobium ciceri was used with seeds at sowing in about half seeds. Inoculation of R. solani, F. solani and M. incognita, chickpea seedlings (two weeks old) were used and inoculations were done Siddiqui et al. . There were 2 treatments i.e. (1) Control; (2) Rhizobium ciceri. Each of these was tested with (I) Control; (II) R. solani; (III) F. solani; (IV) M. incognita (4×2=8). These 8 treatments were tested in two combinations (i) without SiO2 NPS spray (ii) With SiO2 NPs spray (8×2= 16). Each treatment had 5 replications (16×5=80 pots).
Ninety days after inoculation the plants were harvested. Plant growth attributes, photosynthetic pigments, proline and disease index, galling and nematode population were recorded Siddiqui et al., .
Disease index were determined on disease symptoms. Disease rating was done on 0 (no disease) to 5 (severe rot / blight) Nesha and Siddiqui  Estimation of photosynthetic pigments and proline content Photosynthetic pigments (chlorophyll and carotenoid) in the fresh leaf samples were estimated by Mackinney  and proline content by Bates et al. .
The data were analysed through three ways (Rhizobium cicer × SiO2 NPs × Pathogens) analysis of variance (ANOVA) in R (version 2.14.0) statistical software (package- library agricolae). Least significant difference (L.S.D) were calculated at p=0.05. Duncan’s new multiple range test (DNMRT) were employed to denote the significant differences between treatments. Graphs of nematode population and galls per root system were prepared using Sigma Plot™ and error bars showing standard error.
Effects of SiO2 NPs were studied on R. solani and F. solani (Table 1). It had a higher adverse effect on the growth of F. solani followed by R. solani. SiO2 NPs caused 36.25 and 34.60% inhibitions in the growth of F. solani and R. solani respectively (Table 1). SiO2 NPs also had adverse effect on the hatching of M. incognita. It caused 88.37% inhibition in hatching of M. incognita while 43.75% mortality of M. incognita J2 was observed after 48 hours of incubation over control (Table 1).
Table 1:Effect of SiO2 NPs on the growth of R. solani and F. solani in potato dextrose agar medium and on the hatching and mortality of M. incognita.
Three ways ANOVA revealed that individual effect of Rhizobium ciceri, SiO2 NPs and pathogen, and interaction of R. ciceri × SiO2 NPs, Pathogen × R. ciceri, SiO2 NPs × pathogen and R. ciceri × SiO2 NPs × pathogen was significant in proline content and root nodulation at p=0.05 (ANOVA not shown). However, effect of interaction of R. ciceri × SiO2 NPs × pathogen on shoot length, root fresh weight, root dry weight and photosynthetic pigments was non-significant at p=0.05. Similarly, effect of interaction of Pathogen × R. ciceri on root length, shoot fresh weight, shoot dry weight, root dry weight, chlorophyll was also non-significant at p=0.05. Effect of interaction on SiO2 NPs × pathogen on shoot fresh weight, root dry weight and photosynthetic pigments were also non-significant at p=0.05. However, Individual effect of R. ciceri, SiO2 NPs and pathogen and their interactions were significant on other parameters studied (ANOVA not shown).
Inoculation of plants with pathogens under study i. e. M. incognita, F. solani and R. solani resulted in a significant reduction
in plant growth parameters over uninoculated control (Table 2).
Inoculation of M. incognita resulted in a higher reduction (26.07%)
in shoot dry weight followed by R. solani (23.92%) and F. solani
(20.10%). Similarly, inoculation of M. incognita resulted in a
higher reduction (25.42%) in root dry weight followed by R. solani
(23.73%) and F. solani (20.33%). Plants without R. ciceri had poor
nodulation while plants with R. ciceri had higher nodulation (Table
Foliar spray of SiO2 NPs resulted in a significant increase in plant growth parameters over plants with and without pathogen (Table 2). Spray of SiO2 NPs to plants without pathogen caused 25.36% increase in shoot dry weight over uninoculated control. Foliar spray of SiO2 NPs to plants with M. incognita caused 33.01% increase in shoot dry weight while plants with F. solani and R. solani resulted in 39.22% and 39.31% increase in shoot dry weight over plants with respective pathogen alone (Table 3). Foliar spray of SiO2 NPs caused 38.98% increase in root dry weight over uninoculated control. Spray of SiO2 NPs to plants with M. incognita, F. solani and R. solani resulted in 45.45%, 53.19% and 53.33% increase in root dry weight respectively. Nodulation in plants without R. ciceri remain unaffected by inoculation of pathogens and SiO2 NPs spray because of very poor nodulation (Table 2).
Table 2:Effects of SiO2 NPs and Rhizobium ciceri alone and both together on F. solani, R. solani and M. incognita and on the growth attributes and nodulation of chickpea.Values within a column followed by the same letter are not significantly different at p=0.05 by DNMRT.
Table 3: Effects of SiO2 NPs and Rhizobium ciceri alone and both together on F. solani, R. solani and M. incognita and on the photosynthetic pigments (chlorophyll and carotenoid) proline content and disease index of chickpea.
Values within a column followed by the same letter are not significantly different at p=0.05 by DNMRT.
Inoculation of R. solani resulted in a significant increase in plant
growth parameters over pathogen inoculated and un-inoculated
control (except shoot length in uninoculated plants) (Table2).
Inoculation of R. solani resulted in 25.84% increase in shoot dry
weight over uninoculated control. Use of R. solani to plants with M.
incognita, F. solani and R. solani resulted in 40.78, 50.30 and 45.28%
increase in shoot dry weight respectively over their respective
inoculated control (Table 2). Inoculation of R. solani caused 44.06%
increase in root dry weight over uninoculated control. Similarly,
chickpea plants grown with R. solani and with M. incognita,F. solani
and R. solaniresulted in 59.09, 68.09 and 66.67% increases in root
dry weight over respective pathogen inoculated plants. High root
nodulation was observed in plants with R. solani while pathogens
under study had adverse effect on root nodulation caused by R. solani (Table 2).
Foliar spray of SiO2 NPs together with R. solani caused a highest increase in plant growth parameters over both un-inoculated and pathogen inoculated control (Table 2). Spray of SiO2 NPs with R. solani resulted in 109.80% increase in shoot dry weight over control. Similarly, spray of SiO2 NPs plus inoculation of R. solani to plants with M. incognita,F. solani and R. solani caused 139.92, 137.13 and 133.96% increase in shoot dry weight over their respective inoculated control (Table 2). Use of R. solani with SiO2NPs resulted in 120.33% increase in root dry weight over control. However, spray of SiO2NPs to plants with R. solani and test pathogens i.e. M. incognita,F. solani and R. solani resulted in 138.64, 142.55 and 142.22% increase in root dry weight over respective control. Pathogen under study had adverse effect on root nodulation caused by with R. solani and spray of SiO2 NPs also adversely affect root nodulation (Table 2).
Inoculation of plants with M. incognita, and R. solani caused a significant reduction in photosynthetic pigments i. e. chlorophyll and carotenoid contents over uninoculated plants (Table 3). Foliar spray of SiO2 to plants with pathogens under study caused a significant increase in photosynthetic pigments over plants with pathogen alone. Foliar application of SiO2 caused 19.19 and 30.37% increases in chlorophyll and carotenoid respectively over control. Similarly, foliar spray of SiO2 to plants with M. incognita, R. solani and F. solani resulted in 21.50 to 47.79% increase in chlorophyll and 29.21 to 37.13% increase in carotenoid over respective inoculated control. Inoculation of R. ciceri caused 30.80 and 48.44% increase in chlorophyll and carotenoid respectively over control. Similarly, R. ciceri inoculation to plants with M. incognita, R. solani and F. solani resulted in 42.53 to 74.26% increase in chlorophyll and 46.88 to 78.24% increase in carotenoid contents. Combined use of R. ciceri together with SiO2 NPs caused 45.45 and 66.96% increase in chlorophyll and carotenoid respectively over control. Similarly, use of R. ciceri with SiO2 NPs to plants with M. incognita R. solani and F. solani caused 53.76 to 94.11% increase in chlorophyll and 65.47 to 103.99% increase in carotenoid contents (Table 3).
A significant increase in proline contents was observed in plants with M. incognita, R. solani and R. ciceri over control (Table 3). Plants with M. incognita, caused a higher increase in proline contents than by R. solani. Foliar spray of SiO2 or inoculation of R. ciceri also increased proline contents. Use of SiO2 NPs together with R. ciceri resulted in highest increase in proline contents over other treatments (Table 3).
Wet root rot and black root rot disease indices were found 4 in plants with R. ciceri and R. solani respectively (Table 3). Disease indices were 3 in plants with R. solani / R. solani and sprayed with SiO2 NPs or inoculated with R. ciceri. Combined use of SiO2 NPs with R. ciceri reduced disease indices to 2 (Table 3).
Inoculation of M. incognita inoculated alone resulted in high root galling and nematode population (Figure 1, 2). Foliar spray of SiO2NPs or inoculation of R. ciceri reduced galling and nematode population. SiO2NPs spray caused a higher reduction in galling and nematode population as compared to R. ciceri inoculation. Spray of SiO2NPs together with R. ciceri caused highest reduction in nematode population and galling (Figure 1, 2).
Figure 1: Effect of SiO2 NPs (NPs) and Rhizobium ciceri (R) on the population of M. incognita (M) on chickpea.
Figure 2: Effect of SiO2 NPs (NPs) and Rhizobium ciceri (R) on the galling of M. incognita (M) on chickpea.
SiO2 NPs showed antifungal effect in PDA medium because
growth of R. ciceri, and F. solani was inhibited. Similarly, Akpinar
et al.  found that SiO2 NPs resulted in maximum inhibition of F. oxysporum f. sp. radicis lycopersici. SiO2 NPs also showed adverse
effect on M. incognita J2 hatching. Observation of hatched J2 showed
straight body shape and intake of NPs in J2 of M. incognita within
48 h of incubation. Silica nanoparticles caused degeneration of
reproductive organs of nematodes Pluskota et al. . The silicon
carbide nanoparticles had lethality as dead nematodes exhibited
black internal organs Al Banna et al. . It is possible that SiO2
NPs taken by the nematodes were translocated to primary and
secondary organs Wu et al. ; Al Banna et al.  resulting in
deformation of J2 of M. incognita.
In pot experiment, foliar spray of SiO2 NPs increased growth, chlorophyll and carotenoids contents over uninoculated control. Application of Si in plants results in its accumulation and exerts various beneficial effects on plants Liang et al. . Absorbed Si alleviates various abiotic stresses Wang et al. . Si contributes to overcome stresses also may improves plant’s mechanical and physiological properties Epstein ; Ma and Yamaji . Increased growth of plants without pathogen (uninoculated control) by SiO2 application can be due to above reasons.
Foliar spray of SiO2 NPs increased growth attributes of plants
with pathogen. Applications of Si provide resistance against
diseases Fauteux et al. ; Marschner . Various mechanisms are
involved plant disease resistance i.e. defense barriers after pathogen
infection, activation enzymes (defense-related), production of
antimicrobial compounds and regulation of genes Wang et al. .
Si mediated resistance against pathogens was associated with the
changes in activity of soil enzyme and soil microorganisms Wang
et al. . Silicon amendment also reduced disease incidence of
tomato Diogo and Wydra .
Foliar spray of SiO2 can prevent pathogen penetration by formation of a cuticle-Si double layer and decrease disease incidence Ma and Yamaji . Most Si improves mechanical properties and regeneration by cross-linking with hemicellulose in cell walls Guerriero et al. .
Proline contents were found to be increased by the spray of SiO2 NPs in this study. Proline is known to confer tolerance to plants Lehmann et al. . It is a multi-functional amino acid and accumulation of proline acts as a major stress response against pathogens Grote et al. . A correlation between increase in proline contents and increase defense response against pathogens Cecchini et al.  has been observed. Increased proline content was also observed in Arabidopsis thaliana Verslues and Sharma  in plant defense against pathogen. Therefore, in this study increase in proline contents in plants with pathogen was also observed.
Disease indices were found 4 in plants with fungal pathogens viz. R. solani and F. solani. Foliar spray of SiO2 reduced disease indices to 2. Reduction in disease indices by application of SiO2 also confirms antifungal nature of SiO2. SiO2 NPs demonstrated its nematicidal activity by reduction in galling and nematode population. R. ciceri also showed antifungal and nematicidal activity by reducing disease indices, galling and M. incognita population therefore, increase in plant growth. Rhizobial strains have biocontrol properties and can lead to potential control Gopalkrishnan et al. . Rhizobium enhanced resistance level against pathogen by inducing changes in seed proteome and metabolome Sistani et al. . Foliar application of SiO2 NPs had mild adverse effect on rhizobial root nodulation while combined application of NPs and R. ciceri reduce disease severity. This may be possible because of least concentration NPs used as foliar spray. The influence of broad-spectrum of even pesticides and their tolerance to the rhizobia are reported Singh and Wright . It is possible that the R. ciceri used in the present study may have tolerance to SiO2 NPs thereby resultant increase in growth attributes, chlorophyll, carotenoid and proline contents.
SiO2 NPs had displayed antifungal and nematicidal activities towards tested fungi and root knot nematode. Similarly, R. ciceri also provided biocontrol of M. incognita and root rot fungi. Use of SiO2 NPs with R. ciceri was better for the management of pathogens under study than their use alone. Therefore, researches on characterizing of resistant rhizobia are needed so they can be used for the management of chickpea diseases along with SiO2 NPs.
Necessary facilities provided by Aligarh Muslim University Aligarh, India are thankfully acknowledged.
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