Effect of Inoculation of Plant Growth Promoting Rhizobacteria (PGPR) Mix I Formulations on Plant Growth, Yield, Disease Incidence and Disease Severity of Rhizoctonia Leaf Blight of Amaranthus (Amaranthus tricolor L.)

Amaranthus is the most popular and commercially cultivated leafy vegetable in the Southern part of India, especially Tamil Nadu and Kerala. Amaranthus (Amaranthus tricolor L.), belonging to the family Amaranthaceae is a highly nutritious leafy vegetable and is hence known as ‘poor man’s spinach. The leaves are rich source of proteins, vitamins, minerals and dietary fibre.

Plant Growth Promoting Rhizobacteria (PGPR) are a group of bacteria that colonizes plant roots and enhances plant growth and yield by producing plant growth promoting substances. Bacteria of diverse genera were identified as PGPR of which Bacillus and Pseudomonas spp. are predominant. PGPR exert a direct effect on plant growth by production of phytohormones, solubilization of inorganic phosphates, increased iron nutrition through iron-chelating siderophores and volatile compounds that affect the plant signalling pathways. Cassan et al., (2014) discussed about the biosynthesis, metabolism, regulation, physiological role and agronomical impact of phytohormones produced by the model plant-growth-promoting rhizobacteria (PGPR) belonging to the genus Azospirillum, considered to be one of the most representative PGPR.  Lenin and Jayanthi (2012) revealed that, among the plant growth promoting bacteria, P. fluorescens CRPS2 secreted highest amount of both catechol and salicylate type of siderophores followed by Bacillus, Azospirillum and Azotobacter isolates. Additionally, by antibiosis, competition for space and nutrients and induction of systemic resistance in plants against a broad-spectrum of root and foliar pathogens, PGPR reduce the populations of root pathogens and other deleterious microorganisms in the rhizosphere, thus benefiting the plant growth. Nair and Anith (2009) reported reduction in disease severity due to PGPR induced systemic resistance against R. solani in a susceptible amaranth variety, ‘Arun’.

Amaranthus is susceptible to a number of diseases. Among the different diseases affecting amaranth, foliar blight caused by Rhizoctonia solani Kuhn, is considered as the most serious disease in Kerala (Nayar et al., 1996). Rhizoctonia solani Kühn (teleomorph: Thanatephorus cucumeris [A.B. Frank] Donk.) is a soil-borne fungus that causes disease on many economically important crop plants worldwide. The disease which is characterised by light cream coloured spots on the foliage rapidly spreads causing extensive damage leading to very high economic losses. Gokulapalan et al. (2000) observed that the symptoms of the disease were manifested as small irregular whitish cream spots on leaves which enlarged under high humidity to cause extensive translucent and light green lesion and shot hole symptoms. Nayar et al., (1996) reported that on the under surface of the infected leaves, white powdery mass of basidiospores of the telemorph of the causative fungus Thanatephorus cucumeris (Frank) Donk are clearly visible. Control of the pathogen is difficult because of its ecological behaviour, extreme broad host range and the high survival rate of sclerotia under various environmental conditions (Anderson, 1982 and Ogoshi, 1987).

Susceptibility of popular cultivars and humid conditions in Kerala make the disease a serious constraint to tackle for amaranth farmers. The pathogen infects more than 90% of plants in the field and causes considerable economic loss owing to reduced marketability of the produce. Although chemical control of the disease through the use of fungicides can lessen the severity of this aerial blight disease (Gokulapalan et al., 1999), application of chemicals on a regular basis causes serious health hazards. Even though the application of Mancozeb is a promising management measure, the fungicide will causes certain problems if the residue remains on crops, especially in a leafy vegetable like amaranth. Moreover, mancozeb belongs to the group ethylene bisdithiocarbamate whose degradation product ethylene thiourea is a suspected carcinogen (Smitha, 2000) and hence the time and quantity of application becomes crucial. To avoid pesticide residues in agricultural products, alternative methods for the management of pests and diseases using non-hazardous, eco-friendly measures are being explored. The increasing awareness among customers and premium price availability for farmers is shifting the trend in agriculture towards organic farming. Thus, in recent years more research is being carried out for disease management using bio-control agents, which are environmentally safe and are a promising alternative to chemical fungicides. Recognizing the potentiality of organic agriculture and the importance of leaf blight of amaranth in Kerala, a study on the effect of PGPR mix I formulations on growth, yield, disease incidence and severity of Rhizoctonia leaf blight amaranthus was taken up.

The study was conducted at the Onattukara Regional Agricultural Station, Kayamkulam, Alappuzha, Kerala during December 2019 to February 2020. The crop was not supplied with any nutrition other than the PGPR mix I. All the other cultural operations were followed as per the Package of Practices, Kerala Agricultural University. The experiment was conducted in RBD with four replications. Variety Arun (Red) was raised in plots of size 2 x 2 m². The treatments were T₁- Root dip with talc based formulation of PGPR mix I (2%) followed by drenching with talc based formulation of PGPR mix I (2%) at 15, 30 and 45 DAT, T₂- Root dip with talc based formulation of PGPR mix I (5%) followed by drenching with talc based formulation of PGPR Mix I (5%) at 15, 30 and 45 DAT, T₃- Root dip with liquid formulation of PGPR mix I (2%) followed by drenching with liquid formulation of PGPR mix I (2%) at 15, 30 and 45 DAT, T₄- Root dip with liquid formulation of PGPR mix I (5%) followed by drenching with liquid formulation of PGPR mix I (5%) at 15, 30 and 45 DAT and T₅- Absolute control.

The seedlings were raised in nursery beds and were transplanted after 21 days as per treatments by maintaining a population of 70 plants per plot. Observations on yield and various yield parameters viz. number of leaves per plant, plant height, number of branches per plant were recorded at 15, 30 and 45 days after transplanting. Days to flowering and number of plants flowered per plot were also noted to give an indication of the extent of vegetative growth. The response of the plants towards the leaf blight disease under natural epiphytotic condition was evaluated as disease incidence and disease severity. The percent incidence of disease was recorded as number of plants diseased/ total number of plants assessed x 100. Disease severity from each treatment was recorded at 15 days interval on ten randomly selected leaves (three from top, four from middle and three from bottom) from each of five randomly selected plants. Each leaf was scored using a 0-9 scale (KAU, 1996), as given below.
0      no disease
1      1 to 10% infected leaf area
3      >10 to 25% infected leaf area
5      >25 to 50% infected leaf area
7      >50-75% infected leaf area
9      >75% leaf area infected

In addition, percent disease severity or Percentage Disease Index (PDI) was calculated as given below (Wheeler, 1969). All the data recorded were statistically analysed.

Growth characters
 
The height of the plant increased with age of the plant. At all growth stages the tallest plants were recorded for plants from absolute control plot. The shortest plants were recorded from the plots given T₂ (15 DAT), T₁ (30 DAT) and T₃ (45 DAT). At 15 DAT, the maximum number of leaves was produced in T₃ when the plants were subjected to seedling root dip with 2% liquid formulation followed by drenching with 2% liquid formulation at 15, 30 and 45 days of transplanting. At 30 DAT and 45 DAT, the number of leaves produced were maximum when the plants were given a root dip with talc based formulation of PGPR mix I (5%) followed by drenching with talc based formulation of PGPR mix I (5%) at 15, 30 and 45 DAT (Table 1).



The lowest leaf number was recorded by the control plot during the entire period of study. PGPR mix I is a consortium of biofertilizers which can enhance NPK uptake by plants. It was also found to impart disease resistance to vegetable seedlings mainly through improvement in growth (Soumya et al., 2020). A significant enhancement in leaves was observed in plants which were treated with a combination of Pseudomonas consortium, 50% fertilizer and micronutrients. The application of PGPR mix I did not have any significant influence on the number of branches per plant throughout the study. However more number of branches were produced by plants from the control plots (T₅) at 15 days after transplanting and at 30 and 45 DAT, the plants treated with 5% talc formulation of PGPR mix I produced more number of branches (17.05 and 18.05 respectively). Observation from Table 1 indicate that the days needed for flowering was not at all influenced by any of the treatments as there was no significant difference among them. On an average the plants flowered between 45^th and 50^th days under the study. However, Bandopadhyay (2015) had reported that flowering of amaranthus occurred after 21 days of dual inoculation of plant growth promoting rhizobacteria.

Maximum yield was produced from T₂ (1.193 kg m^-2) when the plants were treated with 5% talc formulation of PGPR mix I. But this was found to be on par with T₁ and T₃ (2% talc and 2% liquid formulation respectively). Thus economic yield in amaranthus is possible even with 2% formulation (talc or liquid). This is in accordance with the studies of Gopi and Meenakumari (2020) who revealed that liquid formulation of PGPR mix I is equally effective as talc based formulation of PGPR mix I in enhancing yield and other biometric parameters of amaranthus. The lowest yield was obtained from the treatment with 5% liquid formulation of PGPR mix I (T₄- 0.62 kg m^-2).
 
Disease incidence
 
The field experiment revealed that observations on disease incidence and disease severity exhibited significant differences between the treatments (Table 2).



In plots that received the treatments, number of days taken for first appearance of Rhizoctonia leaf blight symptom in amaranthus ranged from 26 to 35 days after transplanting. But for the plants in the plot T₅ (absolute control), at the time of observation (15 DAT) there was 4.37% disease incidence which means that the symptoms of the disease had appeared much earlier. This is in agreement with the findings of Gireesh and Radhakrishnan (2016) who reported that the number of days taken for first symptom appearance of Rhizoctonia leaf blight in amaranthus plots ranged from 13 to 14 days after transplanting. Throughout the period of study the percentage of disease incidence was found to be the least in T₁ and T₃ when the seedlings were given a root dip followed by drenching with 2% solution using talc or liquid formulations. Thus regardless of talc or liquid formulation of PGPR mix I (2%) seedling dip and drenching at 15, 30 and 45 DAT  provide the least disease expression in amaranthus at Onattukara condition. Even at 45 DAT percentage disease incidence was found to be very less in T1 and T3 (< 2% incidence) compared to plants from absolute plot where there was 9.79% disease incidence. Similar reports of reduced incidence of leaf blight in  A. tricolor with PGPR mix I was reported earlier by Nair and Anith (2009). They revealed that a native isolate, P. fluorescens PN026R was particularly effective in suppressing the disease and promoting plant growth. Uppala et al. (2010) isolated 63 endophytes and evaluated their effect against R. solani and observed that six endophytic bacteria and one endophytic fungus were antagonistic against the pathogen. Azotobacter chroococcum, Azospirillum sp. and Gluconoacetobacter diazotropicus were found to be inhibitory against R. solani in cotton and rice and F. oxysporum in tomato. (Chauhan et al., 2012). They also reported the production of antifungal substances by A. chroococcum.

The trend followed in disease incidence was continued in the percentage of disease severity also (Table 2). The least disease severity percentage was recorded in plants from T₁ plots throughout the study. Even at 45 DAT, the plants expressed only 3.6% disease severity. The next best was from T₃ when the plants did not express any symptom till 30 DAT, but recorded 15.88% at 45 DAT. The treatment T₄ was found to be on a par with T₃ at 15 DAT and at 45 DAT. The highest disease severity was recorded from T₅ (absolute control) which recorded maximum damage compared to other treatments at all stages of crop growth. At harvest more than half the population from T₅ was damaged due to diseased leaves and were found to be unmarketable. This suggests that a seedling dip followed by drenching of 2% PGPR mix I formulation (regardless of talc or liquid) is sufficient to provide desirable disease suppression in amaranthus at Onattukara condition.

The results of the study reveal that amaranthus is greatly influenced by the application of PGPR mix I treatments. Even though taller plants were produced by the plots which did not receive any treatments, maximum number of leaves and number of branches per plant were produced by the plants that were subjected to seedling root dip with 5% talc formulation followed by drenching with 5% talc solution at 30 DAT and 45 DAT. Days to flowering was not influenced by any of the treatments. The highest yield was obtained by seedling dip of 5% talc formulation followed by drenching with the same at 15, 30 and 45 DAT. But yield was not much affected even when the dosage was reduced to 2% formulation (talc or liquid) suggesting the sufficiency of 2% dosage for profitable yielding in amaranthus. In plots that received the treatments took about a month to develop the first disease symptom. Regardless of talc or liquid formulation of PGPR mix I (2%) seedling dip followed by drenching at 15, 30 and 45 DAT provided the least disease incidence and disease severity in amaranthus at Onattukara condition. The plants that did not receive any treatment expressed disease symptoms even before 15 days after transplanting. Hence it can be concluded that use of PGPR Mix I is a prerequisite for effective growth, yield and management of leaf blight of amaranthus at Onattukara.

All authors declare that they have no conflict of interest.