On M. micrantha, the initial symptoms of rust were apparent 5–7 days after inoculations, as chlorotic spots on leaves, petioles and along the stem. Within 12–15 days, the infection developed into dark orange-coloured telia with teliospores embedded in sori.
All the 12 populations of M. micrantha from the group of Andaman islands within the Union Territory of Andaman and Nicobar islands were found to be susceptible to the Peruvian pathotype, with the maximum mean pathogenicity score of 3.88 (Nayashahar) and the minimum of 3.50 (CARI-3) (Table 1). The rust pathotype produced normal life cycle on all the Andaman weed populations (Fig. 2).
The 15 populations of M. micrantha from Assam were found to be susceptible to the rust. Except Sepon, Silchar and Tezpur populations, the other 12 scored the maximum (i.e. PS 4) in pathogenicity (Table 1). The rust pathotype produced normal life cycle on the populations from Assam (Fig. 3). In contrast, there was remarkable variability in susceptibility of Assam M. micrantha plants towards the Trinidadian pathotype of the same rust (Sreerama Kumar et al. 2016). Even the M. micrantha populations from Karbi Anglong and Tinsukia that showed resistance to the Trinidadian pathotype were completely susceptible to the rust from Peru, with a median pathogenicity score of 4. In 11 populations, more than 50% of the leaf/petiole was covered with rust pustules. Fusion of pustules was seen only in Assam populations, specifically in those from Jorhat, Kokrajhar and Silapathar. Such fusion was not observed with the Trinidadian pathotype of P. spegazzinii (Sreerama Kumar et al. 2016). A comparative analysis at CABI (UK) indicated that hyperplasic canker production was high by the Peruvian pathotype but none by the pathotype from Trinidad (Ellison et al. 2008). Besides, the Peruvian pathotype showed a higher systemic spread than the Trinidadian pathotype. Substantial variation in aggressiveness of different P. spegazzinii isolates was also observed by Day et al. (2013a) in M. micrantha accessions from Fiji and Papua New Guinea (PNG).
All the three populations of M. micrantha from Kerala were found to be susceptible to the Peruvian pathotype with the mean pathogenicity scores of 3.90 (Chimmoni), 3.89 (Peechi) and 3.85 (Vazhachal) (Table 1). The pathogen produced normal life cycle on all the weed populations from Kerala (Fig. 4).
Twenty-five plant species, other than sunflower, belonging to ten tribes in the Asteraceae were found to be immune to the Peruvian pathotype of P. spegazzinii (Table 2). On the other hand, the pathogen formed normal pustules on all the positive controls.
In earlier studies with the Trinidadian pathotype, none of the 34 Asteraceae species (other than sunflower), including the present 25, reacted to the rust (Sreerama Kumar et al. 2016).
Fourteen out of the 31 sunflower cultivars/ accessions tested against P. spegazzinii (Peruvian pathotype) showed mild chlorotic flecks (PS 1) (Table 3) on a few top leaves that were directly below the heavy inoculum inside the dew chamber, around 6–8 days after inoculation. Out of those 14 plants, four had produced similar cholorotic flecks (Table 3) with the Trinidadian pathotype (Sreerama Kumar et al., 2016). On the other hand, six accessions that reacted against the Peruvian pathotype had not reacted to the Trinidadian pathotype, and three vice versa (Sreerama Kumar et al. 2016). Although EC-68414, a sunflower accession positive to the Trinidadian pathotype (Sreerama Kumar et al. 2016), was left out this time, four additional accessions were included and all produced chlorotic flecks with the Peruvian pathotype (Table 3). As expected, however, there was no sporulation in any of the cultivars/accessions. When microscopically examined, no mycelial growth was observed in the leaf tissue of the plants showing chlorotic flecks. The inoculated sunflower plants grew and flowered normally. This specific screening precluded the risk of the alien rust fungus posing any threat to sunflower cultivation in India. Ellison et al. (2008) also observed similar chlorotic flecks on sunflower in the primary host-range screening with the Trinidadian pathotype of P. spegazzinii in the UK. Observations till senescence indicated no mycelial growth in the leaves that exhibited chlorotic flecks.
Since ensuring host-specificity is paramount before releasing a biological agent into a new environment, P. spegazzinii (from Trinidad) was earlier rigorously tested and found to be safe to 55 economically important plant species in 31 families in India (Sreerama Kumar et al. 2016). After getting permission from the Plant Protection Advisor to the Government of India, the rust was first released in Assam in October 2005. The pathogen was released in Kerala initially in agricultural systems in August 2006, followed by forest sites. Kerala releases indicated potential, as the fungus was found to have spread to the native population of the weed at all sites. Field persistence, however, was observed only till December. Thereafter, microclimate at the sites was no longer appropriate for pathogen survival (Sankaran et al. 2008). Contrastingly, in Assam, the rust did not infect the M. micrantha plants in situ, possibly because of plant biotypic variation in susceptibility.
Experiences elsewhere indicate that simultaneous and sustained releases of P. spegazzinii at manifold locations and at appropriate time of the year could yield promising results. For example, between 2008 and 2013, an Ecuadorian pathotype of P. spegazzinii was released at multiple sites in PNG and Fiji. In PNG, from some sites, the rust had spread over 7 km within a year. In Fiji, the rust was established at 20 sites on two islands, i.e. Viti Levu and Vanua Levu (Day et al. 2013b). Continuous field studies in PNG indicated that P. spegazzinii could significantly reduce the growth and density of M. micrantha and offered great potential as a biocontrol agent.