Candidatus Phytoplasma Australasia Associated with Alfalfa Witches’ Broom: Symptomatology, Quantitative Loss, Qualitative Loss and Molecular Characterization

The alfalfa (Medicago sativa L.), a leguminous plant vernacularly known as lucerne, is the most important and widely cultivated fodder crop in temperate and tropical regions worldwide (Khan et al., 2001). The crop is often referred to as the queen of fodder crops because of its higher nutritional quality (crude protein (≈16-25%) with higher digestible vitamins and minerals), biomass, seed yield, persistency under dry conditions with its ability to fix atmosphere nitrogen (Ruckle et al., 2017; Kumar et al., 2021). Several biotic stresses limit the production of alfalfa crop. Among which, fungal pathogens like  Verticillium, Phytophthora, Aphanomyces and phytoplasma are the most important pathogens responsible for significant yield in alfalfa (Alhudaib, 2009). However, little leaf or witches’ broom disease associated with phytoplasma is reported to cause significant economic losses in several countries (Gopurenko et al., 2016).

Alfalfa witches’ broom (AWB) or lucerne little leaf disease caused by seven distinct phytoplasma groups (16Sr I, II, VI, VII, IX, XII) has been observed worldwide (Gopurenko et al., 2016). The important phytoplasma group reported on alfalfa includes ‘Candidatus Phytoplasma asteris’ from Bolivia (Jones et al., 2005); ‘Ca. Phytoplasma aurantifolia’ from Iraq (Al-Kuwaiti et al., 2019); ‘Ca. Phytoplasma australasia’ fromTurkey (Ayvaci et al., 2020); ‘Ca. Phytoplasma trifolii’ reported from Canada (Khadhair et al., 1997); ‘Ca. Phytoplasma fraxini’ from Argentina (Conci et al., 2005); ‘Ca. Phytoplasma phoenicium’ from Italy (Marcone et al., 1997) and ‘Ca. Phytoplasma solani’ from Serbia (Starovic et al., 2012).

In India, the little leaf disease of alfalfa was first identified in Karnataka and documented the association of phytoplasma based on the symptoms (Suryanarayana et al., 1996). However, the study did not establish whether the symptoms observed were due to phytoplasma infection. Further, the effect of phytoplasma infection on leaf quality was substantial. Hence, the effort was made to quantify the qualitative and quantitative loss caused by AWB and to confirm the phytoplasma association, molecular characterization was carried out in the present study.

Sample collection and disease incidence

The infected leaf samples showing witches’ broom and little leaf symptoms were collected from the alfalfa field located in central research farm and technology demonstration block, ICAR-Indian Grassland and Fodder Research Institute, Jhansi (IGFRI-1-25° 52’ N 78° 55’ E and IGFRI-2-25° 30’ N 78° 32’ E), Uttar Pradesh, India during Kharif (July-December 2020). The per cent disease incidence was recorded during the sample collection. In addition, the phytoplasma infected weed samples i.e., Parthenium hysterophorus adjacent to the alfalfa fields collected to detect the presence of phytoplasma. Leaf samples from asymptomatic healthy plants were used as a negative control.

Estimation of quantitative and qualitative loss in the infected plant samples

To determine the quantitative loss of biomass in the infected alfalfa, whole infected and healthy alfalfa plants were collected and recorded plant height (n=25), leaf size (n=25), fresh weight and dry weight (n=10). The important fodder qualitative parameters such as crude protein content (% CP: Van Soest et al., 1991), dry matter content (% DM: AOAC, 1990), acid detergent fibre (% ADF: Van Soest et al., 1991) and neutral detergent fibre (% NDF: Van Soest et al., 1991) of infected and healthy plant samples were analyzed. The loss or gain in biomass and nutrients of alfalfa owing to disease was estimated by comparing it with healthy plants.

PCR amplification and sequencing

The total genomic DNA was extracted from leaves of five symptomatic and one asymptomatic alfalfa and parthenium plant using the CTAB method described by Doyle and Doyle (1990). The DNA from a known phytoplasma infected sample (sesamum) and DNA from asymptomatic plants were used as a positive and negative control, respectively. The detection of phytoplasma was based on PCR amplification of 1.8 and 1.2 kb fragments by using universal primer pair P1/P7 (Deng and Hiruki, 1991; Balol et al., 2021) and nested PCR was conducted using universal primers R16F2n/R16R2, respectively (Gundersen and Lee, 1996). The polymerase chain reaction (PCR) was carried out in a 25 μl reaction mixture and amplification was performed in a Thermal cycler as described by Venkataravanappa et al., (2017). Amplified PCR products were separated electrophoretically on 1.2% agarose gel using a capillary electrophoresis unit. Four confirmed positive (including one sample of parthenium) samples containing phytoplasma DNA were sequenced.

Phylogeny and Virtual RFLP analysis

The nucleotide sequences of the 16Sr RNA gene of alfalfa and parthenium phytoplasma were edited, aligned and assembled using CLC Genomics Workbench 12.0 and sequences were deposited in NCBI GenBank. The reliable reference sequences were retrieved from NCBI GenBank and aligned using the ClustalW program available in MEGA X software. A phylogenetic tree was constructed by MEGA X software using Neighbour-Joining [NJ] method adopting 1000 bootstrap replications. All characters were run unordered and of equal weight and gaps were treated as missing data. Further, R16F2n/R16R2 sequence fragments were subjected to virtual RFLP analysis with the iPhyClassifier software (Zhao et al., 2009).

Disease  symptoms and incidence

The distinctive symptoms incited by phytoplasma include stunted growth, witches' brooms (Fig 1), leaf yellowing followed by proliferation of shoots with short intermodal length and severe reduction in leaf size with short petioles (Fig 2A). Later floral parts were converted into leaf-like structures leads to sterility in the plants (Fig 2B). Jones et al., (2005) observed witches’ broom and little leaf symptoms in phytoplasma infected alfalfa plants at Bolivia. Numerous genetically distant 16Sr RNA phytoplasma groups have been reported as the causal agents of diseases leading to different symptoms in alfalfa, including “yellows” and “witches’ broom.”





The average disease incidence ranged from 8-10% in the infected fields. However, the disease incidence reported by Hosseini et al., (2015) was substantially greater than the current study, which revealed disease incidence ranging from 17.8 to 70% in different provinces of Iran. The parthenium plants infected with phytoplasma in the vicinity of an alfalfa field had identical symptoms such as short proliferation with short intermodal followed by floral deformation (Fig 2C). Yadav et al., (2015) reported the phytoplasma infecting Parthenium hysterophorus shares 99.3% rDNA sequence similarity with ‘Ca. P. aurantifolia’ 16S rDNA II-D.

Quantitative and qualitative losses due to AWB disease

Quantitative parameters includes plant height, fresh weight, dry weight and leaf size were measured. Phytoplasma infected alfalfa plant recorded lower plant height (38.60 cm ≈ -35%), fresh weight (40.36g ≈ -46.89%), dry weight (9.07g ≈ -50.08%) and mean leaf size (0.74x0.39 cm) as compared to healthy plants (59.80 cm, 76.00g, 18.17g and 2.29x1.1 cm, respectively). Qualitative parameters viz., crude protein content in the infected plant was reduced by 21.38%, but DM, ADF and NDF were increased by 0.68%, 33.72% and 13.06%, respectively (Table 1). Similar observations were made by Saxena et al., (2002), who reported that alfalfa weevil reduces the green matter yield up to 1.7 t/ha and crude protein content by 0.3t/ha. The crude protein and hemicelluloses content was reduced by 16.43% and 40.00%, but ADF and NDF were increased by 25% in powdery mildew infected cowpea.



Molecular detection of AWB disease

Five symptomatic alfalfa samples and one parthenium sample suspected for phytoplasma infection yielded expected amplicon sizes of 1.8 kb and 1.2 kb for P1/P7 and R16F2n/R16R2 primer pair, respectively, which confirms the presence of phytoplasma in the infected samples. The 16S rRNA gene PCR products of alfalfa (MW 587833 and MW 585599) and parthenium (ON116143 and ON116489) phytoplasma were sequenced, analyzed and deposited in the NCBI GenBank. Sequence similarity search via Blastn showed that the phytoplasma associated with alfalfa witches’ broom shared 99.6 to 100% with ‘Ca. phytoplasma australasia’ belongs to 16SrII  phytoplasma group viz. Scaevola witches'-broom phytoplasma (AB257291), Alfalfa witches'-broom phytoplasma (KY449416, AB259169), all above sequences belong to ‘Ca. P. australasia’ (16SrII group). It confirms that, Parthenium hysterophorus acts as a reservoir of phytoplasma and helps in survival during the off-season. Ca. Phytoplasma aurantifolia survives in P. hysterophorus and spread through insect vector, Orosius albicinctus and causes witches’ broom disease of P. hysterophorus (Yadav et al., 2015).

Phylogeny and virtual RFLP analysis

The phylogenetic tree was constructed based on the 16S rRNA sequences of alfalfa phytoplasma (IGFRI1 and IGFRI2) and parthenium (IGFRI and IGFRI3) phytoplasma with the corresponding region 16S rRNA sequences of different groups of phytoplasma retrieved from NCBI database (Table 2). Based on the 16S rDNA gene sequence, the present study showed that phytoplasma associated with AWB clustered with ‘Ca. P. australasia’ related strains belong to 16Sr II group (Fig. 3). Similarly, ‘Ca. phytoplasma australasia’ was identified as the causal agent of alfalfa witches’ broom disease in Turkey (Ayvaci et al., 2020; Khan et al., 2001).





In-silico RFLP analysis of the F2nR2 fragment of 16S rDNA sequence of alfalfa phytoplasma using iPhyClassifier indicated that the virtual RFLP pattern derived from the query of the F2nR2 fragment of 16S rRNA sequence of alfalfa phytoplasma was identical (similarity coefficient 1.00) to the reference pattern of 16Sr group II and subgroup D (Y10097). Therefore, based on a similar restriction profile, the alfalfa phytoplasma strains in the present study were classified under the 16SrII-D sub-group (Fig. 4). Similar observations were made by Ayvaci et al., (2020) alfalfa witches’ broom disease caused by Ca. phytoplasma australasia belong to 16Sr group II and sub group D.

The present study provides molecular evidence that ‘Ca. P. australasia’ belonging to 16Sr group II and subgroup D responsible for AWB disease in India. The results of quantitative and qualitative properties provide basic knowledge regarding loss caused by the disease. The symptomatology, detection and virtual restriction analysis lead to a better understanding of the pathogen and its host. It also provides a way to develop a holistic approach for diagnosis and designing novel disease management strategies.

All authors declare that they have no conflict of interest.

The authors gratefully acknowledge the Director, ICAR-Indian Grassland and Fodder Research Institute, Jhansi, Uttar Pradesh, India and Head, Crop Improvement Division for providing research facilities and Head, Plant Animal Relationship, ICAR-IGFRI, Jhansi, India, for qualitative analysis of plant samples.