Dispersion of the soybean root rot by Cycloneda sanguinea (Coleoptera: Coccinellidae)

The dispersion of pathogenic microorganisms consists of the transport of pathogens from their source to inoculate a new host. Agricultural and economic importance of the Soybean root rot justifies studying this disease, especially the role of insects as dispersers. The aim of this study was to evaluate the role of the ladybird beetle, Cycloneda sanguinea Linnaeus (Coleoptera: Coccinellidae) in the dispersion of pathogens that cause Soybean root rot. Three pathogen species, Macrophomina phaseolina (Tassi) (Sphaeropsidales: Botryosphaeriaceae), Fusarium incarnatum-equiseti species complex (FIESC), and F. commune (Skovgaard) O’Donnell & Nirenberg were isolated from the midgut of ladybird beetles and cultured. Macrophomina phaseolina was identified by morphology while for the other two species, DNA was sequenced. The DNA extracted was amplified in the Internal Transcriber Spacer (ITS) region, sequenced and compared to voucher sequences deposited in the GenBank. Sequences of nucleotide ITS1-5.8 S were identified in the regions of rDNA-ITS4 ribosomal DNA. This is the first report of Macrophomina phaseolina (Tassi) (Sphaeropsidales: Botryosphaeriaceae), Fusarium incarnatum-equiseti species complex (FIESC), and F. commune (Skovgaard) O’Donnell & Nirenberg, being dispersed by C. sanguinea in Brazilian soybean fields.

Infectious disease has a major influence on the demography of human, plant, and animal populations. Plant pathogen dispersion is a process of movement of propagules from one host to a new host. In this context, plant pathogens produce a large number of propagules with most of them lost before reaching the host plant. Survival strategies of microorganisms such as hyphae and spores in an insect-vector enable the primary cycle initiation of a disease. Insects transmit some plant diseases and can retain the pathogens in the absence of host plants, contributing to the pathogen's survival 1 . Some microorganisms can multiply, reproduce, and undergo cyclic changes inside an insect vector [1][2][3][4][5] .
Fusarium (Hypocreales: Nectriaceae) species cause rot diseases in different crops in South America. Fusarium incarnatum (Roberge) Saccardo was isolated from soil and aerial plant parts in banana and palm trees. This fungus has been implicated in plant diseases, including walnut canker, kangaroo paw blight of ornamental plants, bean pod and seed rot, reducing sorghum seed germination and seedling growth, melon corky dry rot and storage rot problems in mushrooms, besides being the dominant fungus in pearl millet grain 6 . Fusarium equiseti (Corda) Saccardo is a cosmopolitan soil inhabitant worldwide, mainly in dry areas. Damage caused by this fungus occurs in senescent plant tissue and produces diseases such as the sour cherry tree cankers, rot in pumpkin and cucurbit fruits in contact with soil, and diseases in the date palm 6 . Fusarium commune Skovgaard, O'Donnell & Nirenberg was isolated from soil and pea in Denmark and it is widespread in the Northern hemisphere in different crops such as carnation, corn, carrot, barley, and western white pine 7 . In tropical areas, F. commune was isolated from Dajiao bananas in Guangdong, China and Cavendish bananas in Brazil 8 . Fusarium commune caused damping-off and seedling root rot on soybean in the United States 9 .
The increasing incidence of the soybean root rot in Southern Brazil justifies researches on insects dispersing and spreading inoculum of this disease. The aim of this study was to evaluate the dispersal of fungal root pathogens, by isolating the fungi of C. sanguinea collected in soybean fields, followed by a pathogenicity test in healthy soybean plants.

Molecular fungi identification.
Three pathogen species were isolated from the midgut of C. sanguinea: Macrophomina phaseolina, Fusarium incarnatum-equiseti and F. commune. Fungi were identified based on morphological characteristics of Macrophomina phaseolina (Tassi) Goidanich. Specific identification of the other two species was possible by extracting and sequencing DNA from fungi isolated from the digestive tract of larvae and adults. Sequenced ITS1-5.8S-ITS4 regions of the ribosomal DNA were identified as F. incarnatum-equiseti complex species and F. commune (Fig. 1). GenBank accession numbers are KR082312 and KR082314.
Association of detected pathogens with environmental parameters. The frequency of isolation of the fungal pathogens from the C. sanguinea was correlated with soil parameters and weather data. Macrophomina phaseolina had a positive relationship with Ca, Mg, Al, S, clay, P, K, Cu, Zn, pressure, temperature; and had a negative one with organic matter, pH (water), humidity and dew point. Fusarium incarnatum-equiseti and F. commune were negatively correlated with clay, P, Zn, humidity and rain; and a positive correlation with temperature and solar radiation per year (Table 1).

Pathogenicy tests. The healthy soybean plants inoculated with F. incarnatum-equiseti and F. commune
isolates originating from the gastric cecum (digestive tract) of C. sanguinea larvae and adults showed the following symptoms of root rot: internerval leaf chlorosis, internerval necrosis and seedling damping-off and necrosis in shoots and branches. The F. incarnatum-equiseti and F. commune isolation frequency in soybean roots (12.50 ± 3.30% SD) also reduced the seed germination (average 87.50 ± 33.07% SD), lap diameter (average 0.51 ± 0.20% SD) during plant reproduction phase and, increased leaf (2.00 ± 0.89% SD) and root (1.75 ± 0.68% SD) symptoms. Macrophomina phaseolina was not tested for pathogenicity as it is an established pathogen of soybeans.

Discussion
Fusarium species isolated from the C. sanguinea digestive tract were morpho-physiologically identified and confirmed by molecular analysis of the ITS region of group 99, the F. incarnatum-equiseti species complex 23 . Fusarium incarnatum has a history of taxonomic and nomenclatural disagreements because of its isolate morphological variability 23 . Fusarium equiseti have two forms, macroconidia and microconidia. Macroconidia are strongly septate, sickle-shaped, and thick with a different curvature in the mycelium that is oval 6 . The sexual stage of F. equiseti is given by the specific epithet Gibberella intricans Wollenweber. Fusarium incarnatum also have macroconidia and microconidia, slender, with dorsal curved, and a straighter ventral surface (Fig. 2C). The apical cell is curved and tapers to a point; the basal cell is foot-shaped 24 . Microconidia are pyriform and uniseptate. The sexual or teleomorph stage of this fungus has not been described 6,24 .
Fusarium commune have macroconidia and microconidia. Differing from other Fusarium species, the macroconidia of F. commune are short to medium long (3 to 5 µm), straight to slightly curved, slender, and thin-walled. The apical cell is tapered and curved, and sometimes with a sling hook 25 . Microconidia are oval, elliptical, or kidney shaped, usually aseptate (Fig. 3D). Conidia are produced on short monophialides in false heads, singly or in pairs on the aerial mycelium. The sexual or teleomorph stage of F. commune is unknown 7 .
Macrophomina phaseolina infect roots, stems, leaves, and fruits of various plant species in Brazil. It was first reported infecting beans and soybeans, causing charcoal rot disease with numerous tiny, black, and irregularly shaped microsclerotia (Fig. 3). For this fungus, these microsclerotia are survival structures allowing it to persist for a long time in the soil. Transport of contaminated soil during soil tillage and preparation is the one of the main dispersal methods of M. phaseolina within and between fields, and consequently, this fungus has become a globally important crop pathogen 26,27 . However, M. phaseolina lost its ability to reproduce sexually, or can do it only under special environmental conditions [26][27][28] .
The presence of F. incarnatum-equiseti and F. commune spores and hyphae was confirmed into midgut and mouthparts of C. sanguinea on soybean roots and stems. Similar studies have indicated a symbiotic relationship between F. solani and H. hampei 16  Musca domestica L. (Muscidae: Diptera) did not alter the spores in their physical appearance or ability to germinate 29 . One potential biochemical capability detected in Fusarium isolates that may benefit insect hosts include sterol synthesis, detoxification of (plant or venomous preys) derived allelochemicals and secondary metabolites, and nitrogen scavenging from unconventional substrates, including cyanide and formamide 30 . The presence of Fusarium species in the midgut suggests that C. sanguinea may be harboring this fungus in order to benefit from its metabolic capabilities.
Coccinellids are mycophagous, foraging in patches of asexual spores, may acquire conidial inoculum acting as mechanical vectors of pathogens 31,32 . The fungal spores are attractive as food for coccinellids, and therefore should not survive digestion. However, spores remained viable in the mycophagy Halyziini-powdery mildew system 31,32 . Another system involves a diet of aphids present on apple for the coccinellid Hippodamia convergens Guérin-Méneville carrying the fungal pathogen Discula destructiva Redlin (dogwood anthracnose) excreting viable spores [31][32][33] . In this context, spores Fusarium are found in plant tissues where phytophagous insects can acquire them via the cuticle or midgut, and, consequently, these spores are acquired by C. sanguinea through predation.  The presence of spores and hyphae of F. incarnatum-equiseti and F. commune in C. sanguinea indicates a close interaction between C. sanguinea larvae and the Fusarium complex. The teleomorph stage of F. incarnatum and F. commune is unknown but possibly occurs inside the midgut of larvae of C. sanguinea. The dispersion of M. phaseolina, F. incarnatum-equiseti and F. commune by C. sanguinea possibly starts with larval feeding (mycophagy, phytophagy, and zoophagy), absorbing spores and hyphae, near the soil in soybean stem as a protein and sugar source for this insect. The midgut of C. sanguinea can shelter pathogenic fungi so they may complete the reproductive cycle and disperse intact spores by excretion 34 .
High deficiencies or concentrations of N, P, K and micronutrients, and organic matter occurs in sandy and compacted soils, also linked to low pH reduces plant growth, favoring root rot development 35,36 . Mineral nutrients such as K, Ca, Mg, S, B, Mn and Fe favour healthy plants and avoid the entrance of external pathogens 37 . However, the effect of a nutrient on a particular combination (pathogen-host plant), cannot be generalized because some of them may increase disease severity, while others reduce it, as found here.
The weather factors affect the release and dispersal of Fusarium spores into the environment. A passively-released spore is a particle in a given area and its release from a pustule or conidiophore depends on mechanical force (caused by wind or rain) overcoming cohesive forces. Mechanical force from wind or rain is the main cause for particles adhering to plant surfaces to be removed. The chemicals exuded by splash-dispersed fungi, help spores to adhere to plant surfaces when they dry and also help the spores to be splashed when wet by reducing the surface tension of the water film. Changes in humidity, solar radiation and the atmospheric electric charge have been correlated with Fusarium spore mass release 38 . In this study, the mass release of Fusarium spores was observed after fog in the soybean crops ( Fig. 2A,B). This is the first study to report the presence F. incarnatum-equiseti species complex and F. commune with Fusarium disease symptoms dispersed by C. sanguinea in soybean crops. It is also the first report of M. phaseolina, which causes charcoal rot disease, and F. incarnatum-equiseti species complex, and F. commune simultaneously spread by C. sanguinea. This information contributes to our understanding of the geographical distribution of root phytopathogens in Brazil. Moreover, it brings new findings to the insect dispersing phytopathogens and to improve crop protection measures.

Molecular fungi identification.
Larvae and adults of C. sanguinea were disinfected in ethanol and rinsed in sterile distilled water. A tube in which the beetle was immersed was covered by the barrier film, inverted three times, and sonicated for 2 min at 40 kHz (model 8851-34, Cole Parmer, Vernon Hills, IL); the rinse was repeated with sterile distilled water. The mouthparts, prothorax, cuticle, and midgut were dissected and placed in the Eppendorf tubes with 100 mL of 0.85% saline solution 18 in the laboratory of phytopathology at the Universidade Federal de Santa Maria (UFSM). Then, samples of C. sanguinea in saline solution were added to Petri dishes with 3% PDA (Potato Dextrose Agar) media and incubated in a growth chamber (25 ± 6 °C, 75 ± 5% and a photoperiod 12:12 h [L:D]) for seven days 5 .
Amplification and direct sequencing of fungi ribosomal RNA genes for phylogenetic analysis were conducted 40 . The products amplified were purified by precipitation with polyethylene glycol 41 , sequenced by the chain termination reaction method employing the reagent Big Dye 3.1 (Applied Biosystems), and analyzed in an automated capillary sequencer 3500 L (Applied Biosystems).
Species identification was based on the similarity of GenBank sequence databases. Similar sequences were searched in the GenBank using GenBank's BLAST tool and aligned on the BioEdit program with the ClustalW algorithm. An outgroup was also chosen in the GenBank and aligned together to prepare a phylogenetic dendrogram using the MEGA5 software 42 .
A phylogenetic dendrogram was built for the Fusarium spp. isolates obtained from the midgut of C. sanguinea larvae and adults. Evolutionary distances were calculated and evolutionary history inferred with the Maximum Likelihood Method 43 . The branches show the percentage of trees with the associated taxa clustered together. Initial tree(s) for the heuristic search were obtained automatically. The maximum parsimony method was used when the number of common sites was <100 or lower than one-fourth of the total number of sites. The Bioinformatics Neighbor Joining (BIONJ) method with Maximum Composite Likelihood (MCL) distance matrix was used showing branches with the highest probability (log −1416.3605).
Soil and weather analysis. In the soybean crops, soil was removed and sieved in a 2 mm metal wire mesh (0.8 × 0.4 m). The percentage of clay, organic matter, pH (water), and quantities of P, K, Ca, Mg, S, Al, Zn, B, and Cu from soil samples were analyzed at the "Centro de Ciências Rurais" of the "Universidade Federal de Santa Maria" (Santa Maria, Rio Grande do Sul, Brasil). Temperature (°C), humidity (%), dew point (%), pressure (hPa), wind speed (m/s), radiation (kJ/m²), and rainfall (mm) were obtained from the "Instituto Nacional de Meteorologia" (INMET) (Brazil) for the days that samples were taken.