Antoinette P. Malan

Isolation and identification of entomopathogenic nematodes from citrus orchards in South Africa and their biocontrol potential against false codling moth

A survey was conducted to determine the diversity and frequency of endemic entomopathogenic nematodes (EPN) in citrus orchards in the Western Cape, Eastern Cape and Mpumalanga provinces of South Africa. The main aim of the survey was to obtain nematodes as biological control agents against false codling moth (FCM), Thaumatotibia leucotreta, a key pest of citrus in South Africa. From a total of 202 samples, 35 (17%) tested positive for the presence of EPN. Of these, four isolates (11%) were found to be steinernematids, while 31 (89%) were heterorhabditids. Sequencing and characterisation of the internal transcribed spacer (ITS) region was used to identify all nematode isolates to species level. Morphometrics, morphology and biology of the infective juvenile (IJ) and the first-generation male were used to support molecular identification and characterisation. The Steinernema spp. identified were Steinernema khoisanae, Steinernema yirgalemense and Steinernema citrae. This is the first report of S. yirgalemense in South Africa, while for S. citrae it is the second new steinernematid to be identified from South Africa. Heterorhabditis species identified include Heterorhabditis bacteriophora, Heterorhabditis zealandica and an unknown species of Heterorhabditis. Laboratory bioassays, using 24-well bioassay disks, have shown isolates of all six species found during the survey, to be highly virulent against the last instar of FCM larvae. S. yirgalemense, at a concentration of 50IJs/FCM larva caused 100% mortality and 74% at a concentration of 200IJs/pupa. Using a sand bioassay, S. yirgalemense gave 93% control of cocooned pupae and emerging moths at a concentration of 20IJs/cm(2). This is the first report on the potential use of EPN to control the soil-borne life stages of FCM, which includes larvae, pupae and emerging moths. It was shown that emerging moths were infected with nematodes, which may aid in control and dispersal.

Steinernema khoisanae n. sp. (Rhabditida: Steinernematidae) : a new entomopathogenic nematode from South Africa

A new species of entomopathogenic nematode, Steinernema khoisanae n. sp. is described from South Africa. The new species is characterised by morphometrics of the infective juvenile with body length 1076 μm, narrow body diameter of 33 μm, excretory pore 94 μm from anterior end, tail 85 μm long, a = 33, D% = 68, H% = 57, and E% = 111. The lateral field pattern of the new species is 2, 7, 8, 6, 4 and 2. The male of the first generation can be recognised by the spicule and the gubernaculum shape, excretory pore located posteriorly near the end of the pharynx, D% = 88, and SW% = 199. The first generation female can be recognised by the non-protruding vulva and tail bearing a prominent mucron. Steinernema khoisanae n. sp. is characterised genetically by sequences of the internal transcribed spacers and D2/D3 regions of 28S ribosomal DNA, by composition of their sequences and by numerous unique, derived, nucleotide character states. Phylogenetic trees show that S. khoisanae n. sp. and other members of the S. glaseri-group form a monophyletic assemblage.

Heterorhabditis safricana n. sp. (Rhabditida: Heterorhabditidae), a new entomopathogenic nematode from South Africa

CITATION: Malan, A. P. et al. 2008. Heterorhabditis safricana n. sp. (Rhabditida: Heterorhabditidae) : a new entomopathogenic nematode from South Africa. Nematology, 10(3):381-396, doi:10.1163/156854108783900258.

Key elements in the successful control of diapausing codling moth, Cydia pomonella (Lepidoptera: Tortricidae) in wooden fruit bins with a South African isolate of Heterorhabditis zealandica (Rhabditida: Heterorhabditidae).

Abstract The non-insecticidal control strategies currently being implemented in South African orchards for the control of codling moth, Cydia pomonella (L.) may be hampered by wooden fruit bins being infested with diapausing codling moth larvae, acting as a potential source of re-infestation. Key factors contributing to the success or failure of an entomopathogenic nematode application were investigated using the SF 41 isolate of Heterorhabditis zealandica in laboratory bioassays with wooden minibins. Under operational conditions, an application rate of 100 IJs/mL (LD90=102 IJs/mL) effectively controlled codling moth larvae in these bins, and for further laboratory bioassays, the LD50 value of 18 IJs/mL (≈25 IJs/mL) was identified as the discriminating dosage. Maximum mortality was attained when bins were pre-wet for at least 1 min (>90% RH) and maintained at maximum humidity (>95% RH) post-treatment for at least 3 days (LT90=73 h), to ensure nematode survival and subsequent satisfactory infection of diapausing codling moth larvae. Tarping bins achieved the desired high level of humidity required. Furthermore, adjuvants (specifically Reverseal 10™) also improved an application. The study conclusively illustrated that if all the above-mentioned conditions are met, H. zealandica has the potential to successfully disinfest wooden fruit bins of codling moth.

Susceptibility of the Mediterranean fruit fly (Ceratitis capitata) and the Natal fruit fly (Ceratitis rosa) to entomopathogenic nematodes.

The potential of entomopathogenic nematodes, Heterorhabditis bacteriophora, Heterorhabditis zealandica and Steinernema khoisanae, to infect pupariating larvae, pupae and adults of Ceratitis capitata and Ceratitis rosa was investigated in laboratory bioassays. Pupariating larvae and adult flies were susceptible to nematode infection, with no infection recorded for the pupae. Pupariating larvae of C. capitata were generally more susceptible to infection than those of C. rosa. Significantly more larvae of C. capitata were infected by H. bacteriophora. For C. rosa, highest infectivity of larvae was obtained with H. zealandica. In contrast, adults of both species were highly infected by S. khoisanae.

Potential of South African entomopathogenic nematodes (Heterorhabditidae and Steinernematidae) for control of the citrus mealybug, Planococcus citri (Pseudococcidae)

Planococcus citri, the citrus mealybug, is the most important species of mealybug known to infest citrus in South Africa. Various laboratory bioassays were conducted to determine the potential of entomopathogenic nematodes to control P. citri. Adult female P. citri were screened for susceptibility to six indigenous nematode species. P. citri was found to be most susceptible to Steinernema yirgalemense and Heterorhabditis zealandica, causing 97% and 91% mortality, respectively. The development of nematodes after infecting adult female P. citri showed both H. zealandica and S. yirgalemense were able to complete their life cycles inside the host. Further bioassays illustrated a linear relationship between mealybug mortality and the concentration of nematodes applied, with the highest level of control using a concentration of 80 infective juveniles (IJs)/insect. As nematodes would be used as an above-ground application to control P. citri in citrus orchards, available water is a major limiting factor. Insecticidal activity proved to be dependent on the available surface moisture after nematode application. The water activity (a(w)) bioassay indicated that S. yirgalemense to be two times more tolerant to lower levels of free water, with a(w50)=0.96 and a(w90)=0.99, compared to H. zealandica with a(w50)=0.98 and a(w)90=1.0. After application, nematodes have a limited time frame in which to locate and infect hosts, as the level of available free water gradually decreases, as trees dry out. S. yirgalemense proved able to locate and infect P. citri quicker than H. zealandica. Nematode activity was not significantly affected when exposed to 15°C, 20°C and 25°C. IJs were able to infect P. citri at an exposure time as short as half an hour. Results also showed that the first 2-4h post application is the most decisive time for establishing successful infection of mealybugs. This is the first report on the potential use of nematodes for the control of P. citri.

Photorhabdus heterorhabditis sp. nov., a symbiont of the entomopathogenic nematode Heterorhabditis zealandica.

The bacterial symbionts SF41T and SF783 were isolated from populations of the insect pathogenic nematode Heterorhabditis zealandica collected in South Africa. Both strains were closely related to strain Q614 isolated from a population of Heterorhabditis sp. collected from soil in Australia in the 1980s. Sequence analysis based on a multigene approach, DNA-DNA hybridization data and phenotypic traits showed that strains SF41T, SF783 and Q614 belong to the same species of the genus Photorhabdus with Photorhabdus temperata subsp. cinerea as the most closely related taxon (DNA-DNA hybridization value of 68%). Moreover, the phylogenetic position of Photorhabdus temperata subsp. cinerea DSM 19724T initially determined using the gyrB sequences, was reconsidered in the light of the data obtained by our multigene approach and DNA-DNA hybridization experiments. Strains SF41T, SF783 and Q614 represent a novel species of the genus Photorhabdus, for which the name Photorhabdus heterorhabditis sp. nov. is proposed (type strain SF41T=ATCC BAA-2479T=DSM 25263T).

Heterorhabditis noenieputensis n. sp. (Rhabditida: Heterorhabditidae), a new entomopathogenic nematode from South Africa

A new entomopathogenic nematode in the genus Heterorhabditis is described from South Africa, from two singular isolates found 1000 km from each other, from beneath a fig tree and in a citrus orchard, respectively. Morphological and molecular studies indicate both isolates to be the same and a new undescribed Heterorhabditis species. Comparison of sequences of the internal transcribed spacer (ITS) rDNA and the D2D3 region of the 28S rDNA gene with available sequences of other described species within the genus, indicate the two isolates as a new species. Phylogenetic analysis of the sequence data concerned placed the new species, H. noenieputensis n. sp., closest to H. indica and H. gerrardi in the indica-group. The new species, H. noenieputensis n. sp., is distinguished from other species in the genus by a combination of several morphological traits of the males and the infective juveniles (IJs). The new species differs from all other species previously described, as regards the body length of the IJs, except for H. indica and H. taysearae, in which the IJ is smaller. The IJ also differs from that of H. indica in the length of the oesophagus, the body diameter, the length of the tail and the E%. In addition, males of H. noenieputensis n. sp. differ from their closest relative, H. indica, in the position of the excretory pore, SW% and D%; and from H. gerrardi in the length of the oesophagus and SW%. The seventh pair of genital papillae of H. noenieputensis n. sp. are normally developed, while for H. indica they are often branched or swollen at the base, while 8 and 9 are usually absent in both species.

Photorhabdus luminescens subsp. noenieputensis subsp. nov., a symbiotic bacterium associated with a novel Heterorhabditis species related to Heterorhabditis indica

The bacterial symbiont AM7(T), isolated from a novel entomopathogenic nematode species of the genus Heterorhabditis, displays the main phenotypic traits of the genus Photorhabdus and is highly pathogenic to Galleria mellonella. Phylogenetic analysis based on a multigene approach (16S rRNA, recA, gyrB, dnaN, gltX and infB) confirmed the classification of isolate AM7(T) within the species Photorhabdus luminescens and revealed its close relatedness to Photorhabdus luminescens subsp. caribbeanensis, P. luminescens subsp. akhurstii and P. luminescens subsp. hainanensis. The five concatenated protein-encoding sequences (4197 nt) of strain AM7(T) revealed 95.8, 95.4 and 94.9 % nucleotide identity to sequences of P. luminescens subsp. caribbeanensis HG29(T), P. luminescens subsp. akhurstii FRG04(T) and P. luminescens subsp. hainanensis C8404(T), respectively. These identity values are less than the threshold of 97 % proposed for classification within one of the existing subspecies of P. luminescens. Unlike other strains described for P. luminescens, strain AM7(T) produces acid from adonitol, sorbitol and xylitol, assimilates xylitol and has no lipase activity on medium containing Tween 20 or 60. Strain AM7(T) is differentiated from P. luminescens subsp. caribbeanensis by the assimilation of N-acetylglucosamine and the absence of haemolytic activity. Unlike P. luminescens subsp. akhurstii, strain AM7(T) does not assimilate mannitol, and it is distinguished from P. luminescens subsp. hainanensis by the assimilation of trehalose and citrate, the inability to produce indole from tryptophan and the presence of acetoin production and urease activity. Strain AM7(T) ( = ATCC BAA-2407(T)  = DSM 25462(T)) belongs to a novel subspecies, and is proposed as the type strain of Photorhabdus luminescens subsp. noenieputensis sp. nov.

Effect of humidity and a superabsorbent polymer formulation on the efficacy of Heterorhabditis zealandica (Rhabditida: Heterorhabditidae) to control codling moth, Cydia pomonella (L.) (Lepidoptera: Tortricidae)

Abstract Adequate moisture levels are required for nematode survival and subsequent efficacy as entomopathogens. Formulation of nematodes aimed at aboveground applications may assist in maintaining such moisture levels. In this study, we report the effects of a superabsorbent polymer formulation, Zeba® on the performance of an entomopathogenic nematode, Heterorhabditis zealandica Poinar, for controlling diapausing codling moth, Cydia pomonella (L.) larvae in cryptic habitats on trees. Water activity (aw-value) on bark was considered to be an indication of moisture levels on trees in cryptic habitats where codling moth larvae are known to occur, thereby influencing nematode efficacy. H. zealandica was only able to infect codling moth larvae at aw≥0.92, with aw50=0.94 and aw90=0.96. Laboratory experiments in which nematode concentration was investigated indicated a positive linear relationship between the concentration of nematodes applied and the level of control obtained, with the highest level of mortality recorded at 80 IJs/larva, requiring at least 4 h of conditions conducive to nematode activity to ensure infectivity and subsequent efficacy. Further experimentation showed that the use of the Zeba formulation, together with the nematodes, improved the level of control obtained at 60% and 80% RH in the laboratory and that it also enhanced the survival and infection-ability of the nematodes in the field. The study conclusively illustrates that the tested formulation assisted in maintaining adequate moisture levels on the application substratum, as required for nematode survival and subsequent efficacy.