Monitoring of the field application of Metarhizium anisopliae in Brazil revealed high molecular diversity of Metarhizium spp in insects, soil and sugarcane roots

Abstract

The use of Metarhizium against sugarcane spittlebugs in Brazil is one of the most successful and long lasting biological control programs using entomopathogenic fungus in the world. However, studies to monitor the fate of this fungus on the sugarcane agroecosystem are rare, especially with respect to its persistence, efficacy in pest control and impact on the local populations of Metarhizium. The present study aimed at documenting the efficacy and persistence of M. anisopliae strain ESALQ1604 in a sugarcane field by using microsatellite molecular markers. The species diversity of Metarhizium was characterized in insects, soil and sugarcane roots in a sprayed and an unsprayed plot. Although the infection rates were not very high (≤ 50%), the applied strain was recovered from spittlebugs after 7, 30 and 60 days' post-application, but accounted for only 50%, 50% and 70.5% of all insects killed by M. anisopliae, respectively. All haplotypes from spittlebug were associated with a single subclade of M. anisopliae. The highest haplotype diversity was found in soil (h = 0.989) and in the smallest in spittlebug (h = 0.779). Metarhizium robertsii, M. anisopliae, M. brunneum; one taxonomically unassigned lineage was found in soil and only M. brunneum and M. anisopliae were isolated from roots. This study revealed the great diversity of Metarhizium spp. in the sugarcane agroecosystem and the importance of the local population of M. anisopliae on spittlebugs management.

Figure 1

Frequency of the multilocus microsatellite haplotypes (MMH) recovered from infected spittlebugs collected on three dates before fungus application (PA) and seven (7D), 30 (30D) and 60 (60D) days after fungus application. The MMH, 152 represents the applied strain (ESALQ1604).

Figure 2

Percentage of insect infection (left y-axis) in sugarcane fields on three dates before and four dates after ESALQ1604 application, and the number of insects (right y axis) recorded by sample spot (linear meter). Percentage of fungus infection in adults and nymphs (bars) and the average of insects collected by sample point per date (dashed rows). The x-axis shows the sampling date and the days before or after the application of the fungus (Date (days before or after application)). Blue stars represent the dates in which ESALQ1604 was recovered from spittlebugs. The arrow represents the moment of application of the fungus ESALQ1604.

Figure 3

Minimum spanning network of 152 multilocus microsatellite haplotypes (MMH) of Metarhizium spp. Circle sizes are proportional to the number of isolates recovered for each MMH. The black arrow indicates the MMH that represent the applied strain (ESALQ1604). The width of lines between circles indicates Rogers’ genetic distance. Different colors indicate the origin of isolates (insect, soil or root).

Figure 4

Maximum likelihood phylogeny of 5′-TEF of 128 multilocus microsatellite genotypes found in this study. A total of 142 Brazilian Metarhizium isolates from the ESALQ culture collection are represented. Black circles represent strains from CBS Culture Collections (n = 2) and from taxonomically validated reference accessioned in ARS Entomopathogenic Fungal Culture Collection (ARSEF, n = 7, two of them representing the clades Mani 2 (ARSEF_6347), Mrob 1 (ARSEF_727)). The black triangle represents strains previous published in Rezende et al. (n = 6) and black square represent a strain (IP 145) previous published in Rocha et al.. Above branches, bootstrap support is shown for ML (>70).

Figure 5

Percentage of Metarhizium species addressed to the haplotypes representatives isolates from soil (n = 113), roots (n = 6) and insects (n = 14) from sugarcane field.

Figure 6

Tests of recombination for M. anisopliae (Mani 2), n = 14 (a) and M. robertsii (M rob 1) n = 56 (b) multilocus genotypes represented by isolates exclusively from soil and insect.