Ricardo Moreira Souza

Efficacy of Heterorhabditis baujardi LPP7 (Nematoda: Rhabditida) applied in Galleria mellonella (Lepidoptera: Pyralidae) insect cadavers to Conotrachelus psidii, (Coleoptera: Curculionidae) larvae

Abstract The guava weevil, Conotrachelus psidii, is a major pest of guava in Brazil causing severe reduction in fruit quality. We assessed its susceptibility to Heterhorhabditis baujardi LPP7 infective juveniles (IJs) in the greenhouse and under field conditions applying the nematodes in cadavers of seventh instar Galleria mellonella larvae. Field persistence of these nematodes in the soil was evaluated through G. mellonella-baiting. Insect cadaver concentrations of 2, 4 and 6 applied in pots in the greenhouse experiment caused significant mortality compared to the control. Significance differences were observed in the field between control and treatments only when six cadavers per 0.25 m2 were applied. Infective juveniles from the cadavers persisted 6 weeks after application in the field, but decreased greatly thereafter. Our work demonstrates that H. baujardi LPP7 IJs emerging from G. mellonella cadavers can be efficacious against guava weevil fourth instar larvae. Also, we demonstrated the long-term persistence of IJs in the soil.

Dispersal of Heterorhabditis baujardi LPP7 (Nematoda: Rhabditida) applied to the soil as infected host cadavers

In orchards, in cases where the insect pests are concentrated under the canopy, entomopathogenic nematodes can be applied as infected host cadavers. This study evaluated the distribution of Heterorhabditis baujardi Phan, Subbotin, Nguyen & Moens strain LPP7 applied as one or 15 host cadavers per plot in a guava orchard. The plots were located between guava trees, with the host cadavers being placed in the centre. Soil samples were taken at 1, 3, 5, 8 and 10 weeks after cadaver application. On each date, the plots were sampled at 30, 60, 90 and 120 cm from the cadaver application point and at three different depths (0 – 10, 10 – 20 and 20 – 30 cm). The highest average number of recovered infective juveniles was found at 90 cm from the cadaver application point, and at 10 cm depth and on the fifth week after application. There was no significant effect on the number of recovered infective juvenile nematodes, when they were applied as one or 15 cadavers, although the most uniform dispersal was found when 15 host cadavers were applied. We discuss the significance of these results.

Avaliação de acessos de Psidium spp. quanto à resistência a Meloidogyne enterolobii

Forty-seven accessions of Psidium spp. were evaluated under greenhouse conditions for resistance to M. enterolobii, as part of an effort to develop resistant rootstocks and cultivars. For each accession, five to seven plants were produced from stem cuttings or from true seeds and, at the stage of four pairs of leaves, they were inoculated with 500 nematode eggs. One hundred and thirty-five days later, the plants were removed from the pots and half of root system was processed for extraction of eggs. The plants were replanted with the remaining roots. The egg counts, obtained from three 1 mL aliquots per plant, were multiplied by two to obtain the final nematode population (Pf). The Pf values of all aliquots were submitted to ANOVA, which revealed significant differences among accessions and among plants of the same accession. The plants were classified as resistant or susceptible based on the reproduction factor (RF = Pf/500). All plants of cattley guava (P. cattleyanum) (accessions 115 and 116) were resistant (RF <1) to M. enterolobii, while other Psidium spp. presented some plants with RF below or just above 1. These plants will be propagated and reassessed for resistance to M. enterolobii.

Guava decline: effect of root exudates from Meloidogyne enterolobii-parasitized plants on Fusarium solani in vitro and on growth and development of guava seedlings under controlled conditions

In guava decline, Fusarium solani-immune guava trees become susceptible to extensive root rot caused by this fungus after parasitism by Meloidogyne enterolobii. To understand the mechanisms involved in this disease, root exudates were collected from nematode-inoculated (NI) or uninoculated (UN) guava plants cultivated in sand. After filtration through a Millipore® membrane, NI and UN exudates were used: i) to prepare media to assess their effect on mycelial growth and production of propagules of F. solani isolate UENF/CF 163, and ii) to incubate macro- and microconidia to assess their effect on germination. NI exudates promoted (P < 0.05) more mycelial growth and production of propagules than UN exudates or water. NI and UN exudates were used to water guava seedlings laid over seed germination paper inside plastic boxes. Half of the seedlings had an agar plug colonized by the fungus positioned in the collar region. Upon watering with NI exudates the fungus caused (P < 0.05) extensive rotting of the seedlings’ rootlets. NI and UN exudates, either unlyophilized or lyophilized and re-suspended to the original concentration, were used to water guava seedlings grown in sterile sand before being inoculated (or left uninoculated) as described before. Solely upon watering with NI exudates, in its unlyophilized form or after lyophilization, the fungus caused a reduction (P < 0.05) of shoot and root biomass associated with rotting of roots. These results suggest that M. enterolobii induces chemical changes in the root exudates of guava trees, which are necessary for root invasion causing root rotting by F. solani.

Greenhouse and field assessment of rhizobacteria to control guava decline

In an effort to devise a biological strategy to control guava decline, 120 rhizobacteria isolates were obtained from symptomless guava trees located in Meloidogyne enterolobii-infested orchards. Of those isolates, 44 were assessed for their potential to reduce nematodes reproduction: for each isolate, six guava stem cuttings were embedded for eight hours with bacterial suspension and transplanted. Upon development of the roots, the plants were inoculated with 2000 nematode eggs and allowed to grow for four months under greenhouse. Seedlings embedded with water, inoculated or not with the nematode, served as controls. All treatments were equivalent in the five variables that assessed plant development. Several rhizobacteria reduced (p<0.05) the final nematode population (Fp), Fp/gram of root and reproduction factor, although not to satisfactory levels. Subsequently, a two-year experiment was set up in a guava orchard affected by guava decline, in which three of the most effective rhizobacterial isolates were compared with the biological products Nemat® and Nemaplus® for their ability to reduce variables related to nematode parasitism and increase guava productivity. Seven bimonthly applications of these treatments under the tree canopy were unable to reduce nematode parasitism and increase productivity. The decline and death of some plants forced the experiment to be stopped after the first harvest. In conclusion, rhizobacteria applications seem unable to reduce the parasitism of M. enterolobii on guava plants, and even less to reduce the extensive root decay or alleviate the physiological stress suffered by trees affected by guava decline.

Greenhouse and field assessment of different organic compounds against guava-parasitic Meloidogyne enterolobii

Guava decline is a complex disease involving Meloidogyne enterolobii and Fusarium solani and it has caused major direct losses to Brazilian growers. Although several strategies have been sought to control the nematode, the use of organic soil amendments is currently the best approach to manage this disease. To assess the best amount of meat and bone meal (MBM) to be incorporated into the soil, guava seedlings inoculated with M. enterolobii were treated with 1-5% v/v of the MBM. Ninety days later variables related to nematode reproduction and plant development were evaluated, which indicated a potential nematicidal effect of the MBM at 3%. Another experiment assessed nematode- and plant-related variables 90 days after treatment of the seedlings with MBM, chitosan, shrimp shell or neem cake at 3%, 0.05%, 2% and 0.1% v/v, respectively. The MBM ranked first, reducing nematode reproduction. This MBM rate was converted to 25 kg/tree and assessed in three application regimes (monthly, bimonthly or trimonthly), for six months, in an orchard affected by guava decline. The variables assessed were soil density of colony forming units (CFU) of bacteria and fungus, and soil and/or root density of M. enterolobii, Helicotylenchus sp., and of different nematode trophic groups. In all three application regimes the MBM reduced all plantparasitic nematodes in the soil and the fungus CFUs. It also promoted an increase in bacterial CFU and bacterivorous nematodes.

Fine structure and phylogenetic significance of a muscular basal bulb in Basiria gracilis Thorne, 1969 (Nematoda: Tylenchidae)

Classical hypotheses of the phylogenetic position of Tylenchida within Secernentea are evaluated considering the comparative fine structure of the basal bulb of the pharynx of Basiria gracilis, putatively a representative of the most morphologically conserved members of the order. Unlike other Tylenchida examined in detail, B. gracilis has a full complement of nonglandular cells in the basal bulb including 13 radial muscle cells and six marginal cells. With respect to types and numbers of cells, the basal bulb of B. gracilis most nearly resembles representatives of Cephalobina, differing only by three vs five gland cells. There are more muscle cells in the basal bulb of B. gracilis than in representatives of Rhabditina (ten muscles) and Diplogastrina (six muscles). Considering Teratocephalus as a representative of the outgroup (to establish character polarity), B. gracilis appears to retain most of the conserved features of the basal bulb of Secernentea, with the exception of loss of a heavily cuticularised grinder valve or pump.

Assessing the influence of the entomopathogenic nematode Heterorhabditis baujardi LPP7 (Rhabiditina) on embryogenesis and hatching of the plant-parasitic nematode Meloidogyne mayaguensis (Tylenchina)

Two assays were conducted to assess the influence of infective juveniles (IJs) of Heterorhabditis baujardi LPP7 on the embryogenesis and hatching of Meloidogyne mayaguensis. In the first assay, eggs were incubated in water alone or in the presence of infective juveniles, and completion of embryogenesis was evaluated 14days later. In the second assay, unhatched second-stage juveniles were incubated in distilled water alone or in the presence of infective juveniles. Cumulative hatching was compared at various time intervals. Embryogenesis was not affected, whereas second-stage juveniles hatching was delayed probably because of the eggs permeability to noxious metabolites released by Photorhabdus luminescens, which is the bacterial symbiont of H. baujardi.

Inheritance of resistance to Meloidogyne enterolobii and individual selection in segregating populations of Psidium spp

Interaction between the phytonematode Meloidogyne enterolobii and the fungus Fusarium solani has caused direct and indirect losses in the entire guava production chain and consequent extermination of guava plantations throughout Brazil. The combined action of these two pathogens is known as “guava decline”. In order to obtain and assess Psidium spp. interspecific hybrids for resistance to the nematode M. enterolobii, interspecific crosses of P. guineense (susceptible araçá) x P. cattleyanum (resistant araçá); P.guineense (susceptible araçá) x P. guajava (susceptible guava) and P. cattleyanum (resistant araçá) x P. guajava (susceptible guava) were conducted. These crosses resulted in hybrid immune, susceptible and resistant to Meloidogyne enterolobii. The chi-square test rejected the hypothesis of monogenic inheritance with incomplete dominance, which corroborates that this trait has polygenic action. Predictions of genetic values ​​and parameters were obtained by the REML / BLUP procedure, at individual level. Finally, the 30 selected individuals (immune and resistant) were obtained, which will be backcrossed with guava for the recovery of the agronomic traits desired and subsequent release of a new cultivar.

CONTROLE BIOLÓGICO DA MOSCA-DO-MEDITERRÂNEO Ceratitis capitata UTILIZANDO NEMATOIDES ENTOMOPATOGÊNICOS EM LABORATÓRIO

The present study investigated under laboratory conditions the use of entomopathogenic nematodes strains separately or in combinations, as biological control agent of Mediterranean fruit fly, Ceratitis capitata Wied. (Diptera, Tephritidae). In the first bioassay, eight strains were used separately (Steinernema carpocapsae NCALL, Heterorhabditis bacteriophora HP88, H. baujardi LPP7, H. indica LPP1, H. indica LPP14, H. sp. LPP9, H. sp. LPP17 e H. sp. LPP12). For each treatment, 20 test tubes with sand, 10 larvae of C. capitata and 100 infective juveniles (IJs) diluted in 1 cm 3 of distilled water were used. In the treatment control only 1 cm 3 of distilled water was added. In the second bioassay it was used the same material; however, the number of C. capitata larvae was reduced to five and strains of nematodes combined in pairs, in a total of 100 IJs per replicate (50 individuals of each strain). All treatments were stored in an incubator for 15 days (28 oC, 80% RU and 12 h photoperiod). The average mortality of larvae L3 was evaluated by Tukey test at 1%. The strains H. baujardi LPP7, H. indica LPP14, H. sp. LPP17 and H. sp. LPP12 were the most efficient ones, reaching mortalities range between 75 and 98.5%. In the second experiment, the most effective combinations were H. indica LPP14 + H. sp. LPP9 and H. sp. LPP17 + H. sp. LPP12 with mortality of 60 and 82%, respectively. We concluded that the use of NEPs in the biological control of C. capitata is a feasible alternative either using species separated or in combination, but the first one may reach higher mortality. Key-words: mortality; juveniles; pairs; biocontrole; Tephritidae