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6Russian Journal of Nematology, 2023, 31 (2), 111 - 114
The effect of drying a water suspension of Pasteuria penetrans on spore viability
Emmanuel A. Tzortzakakis
Institute of Olive Tree, Subtropical Crops and Viticulture, Department of Viticulture, Vegetable Crops, Floriculture and Plant Protection, ELGO-DIMITRA, 32A Kastorias Street, Mesa Katsabas, 71307, Heraklion, Crete, Greece
e-mail: [email protected]
Accepted for publication 23 July 2023
Summary. Pasteuria penetrans (Pp) is a mycelial and endospore forming bacterium parasitising root-knot nematodes (Meloidogyne spp.), with potential as a biocontrol agent. It is stored either in the form of ground dried roots containing infected females, or water suspensions deriving from crushed infected females. For its transportation between laboratories the dried root material and the liquid formulation may be blocked by posting regulations. Water suspensions containing Pp spores (Pp3 and Pp blend) were left to dry and remained dried for 2 or 12 months. After hydration, juveniles of M. incognita and M. javanica were exposed in Petri dishes containing spores of Pp blend and Pp3 respectively, either hydrated after the drying period or kept continuously in water suspension (control) and the number of attached spores recorded after 24 h incubation. Spore encumbered juveniles of M. incognita were inoculated on pepper and of M. javanica on tomato. The plants grew for 50 days in a growth room and afterwards the number of egg masses per root was recorded. Females without egg masses that were suspected to be infected were checked for the presence of spores inside. Both spores dried for 2 or 12 months and then hydrated and spores kept continuously wet, attached on juveniles at similar rates, and had a similar efficacy in reducing the number of egg masses and infection rate of females. Therefore, a dried water suspension of Pp can overcome posting restrictions and can be safely used for further multiplication of the parasite. Key words: biocontrol, Meloidogyne javanica, Meloidogyne incognita, nematode parasite, tomato, pepper.
Pasteuria penetrans (Pp) is a mycelial and endospore forming bacterium parasitising root-knot nematodes (Meloidogyne spp.), with biocontrol potential. Spores of Pp are immobile and attach on second-stage juveniles (J2) when they move. The J2 encumbered with spores invades the root and develops while the spores germinate and proliferate inside its body. The development of Pp does not impair feeding, moulting or growth of the nematode, but it selectively destroys the reproductive system of females. The infected female becomes filled with spores and does not lay eggs (Chen & Dickson, 1998). For use as a biocontrol agent, roots containing spore-infected females are thoroughly air-dried to destroy egg masses produced by non-infected females and afterwards the roots are ground. Pp is stored in the form of this dried root material at room temperature (Stirling & Wachtel, 1980). The spores contained in dried roots maintain their ability to attach to J2 and subsequently infect females after being hydrated. Alternatively, infected females can be extracted from fresh roots and crushed in water to release the spores. Therefore, a suspension of Pp
spores, free of any surrounding root material is obtained and can be stored in a refrigerator.
Resistance of Pp spores to desiccation for a brief period, was tested by drying a spore suspension in an open glass Petri dish overnight at 28°C. Afterwards, spores sticking to the glass were re-suspended with a toothbrush in water and designated as 'dried spores' to differentiate them from the original 'wet spores'. Attachment tests indicated, that 'dried spores' adhered to M. incognita much more rapidly and at higher rates than 'wet spores' but their ability to infect was not tested (Netscher & Duponnois, 1998).
The idea for the current work originated from a 'casual' observation, when a spore suspension of Pp kept in a fridge, was by a mistake left to dry for an unknown period, less than 1 year. It was found that after subsequent hydration, spores attached on J2 and infected females. Therefore, two experiments were conducted to investigate whether spores of Pp, derived from squashed females in a water suspension, retain their ability to attach to J2 and infect females of M. javanica and M. incognita after
© Russian Society of Nematologists, 2023; doi: 10.24412/0869-6918-2023-2-111-114
the suspension dries and remains dry for periods of 2 months and 1 year.
MATERIAL AND METHODS
Nematodes and Pasteuria penetrans. Eggs of M. javanica and of M. incognita were collected from roots of tomatoes and peppers grown in pots (Hussey & Barker, 1973) and incubated in extraction dishes for 4 days to obtain J2 for the experiments. Both nematode populations originated from Crete, Greece and had been maintained in pots for several years.
A spore suspension of Pp blend (a blend of six isolates of different continental origins, e.g., Africa, Oceania, USA; Nasiou et al., 2020), with a density of 1.7 * 104 spores ml1, had been produced by squashing spore-infected females of M. javanica. After using it for experiments, a small quantity (ca 6 ml) remaining in the bottom of a small glass beaker, was stored in a refrigerator and kept for reference. The glass beaker had been covered with parafilm which by a mistake was loosely tighten and the suspension progressively dried. The suspension was observed to be dry after storage for 1 year, but the period that the suspension had been dry could not be determined. For spore recovery, 6 ml of distilled water was added to the beaker, vortexed and remained for 2 weeks in a refrigerator, so as to ensure hydration of spores. Afterwards, the suspension was vortexed to dispersing the spores and the density was estimated with a haemocytometer and found to be 104 spores ml1. Hatched J2 of M. javanica were transferred to a 5.5 cm diam. Petri dish and 4 ml of the spore suspension was pipetted inside. After 48 h incubation at 25-28°C, spores were observed on J2 using an inverted microscope at *200. The J2 encumbered with spores were used to inoculate a tomato plant (Solanum lycopersicum 'Ace') grown in 250 ml plastic pot filled with a commercial soil substrate. The plant was kept in a growth room at 24-26°C and 14 h photoperiod for 50 days. Afterwards it was uprooted, the roots washed thoroughly and females without egg masses were selected and extracted from the root under a dissecting microscope. Those females were put in drops of distilled water on glass slides, crushed with cover slips and examined at *400 for the presence of mature spores of Pp. Since infected females were found, it demonstrated that the spores of Pp did not lose their ability to infect after the suspension dried.
The cover slips were removed and the pieces of the squashed females stuck on glass slides and
coverslips were washed in a glass beaker and vortexed to disperse the spores. This fresh suspension of Pp blend was kept stored in a refrigerator for 2 weeks and afterwards was used to prepare 10 ml distilled water dilutions with ca 70,000 spores ml-1 in three glass vials. Similarly, three other vials were prepared with another Pp isolate, called Pp3 (from South Africa; Spaull, 1981), containing 3 ml dilutions with ca 700,000 spores ml-1.
Drying and hydration process of Pasteuria penetrans. The one vial for Pp blend and Pp3 was capped tightly (wet spore treatment) while the other two remained opened (dried spore treatment) and all were placed in an incubator at ca 26°C, for
1 month, until the water in the opened vials had completely evaporated.
Afterwards, the opened vials of both Pp isolates were capped tightly and were put in the refrigerator with the vials containing the aqueous spore suspensions. After a period of 2 months, one empty vial was filled with 10 ml (for Pp blend) or 3 ml (for Pp3) distilled water, vortexed and was placed back to refrigerator, so as to ensure spore hydration. Ten days later, all vials were vortexed to disperse the spores and the spore density was determined. The spore densities were: Pp blend wet spore treatment 75,000 spores ml-1, Pp blend dried spore treatment 57,000 spores ml-1, Pp3 wet spore treatment 1,000,000 spores ml-1 and Pp3 dried spore treatment 765,000 spores ml-1. Twelve months from the beginning of the experiment, water was added in the second set of vials with the dried spores and processed as previously described. The spore densities were: Pp blend wet spore treatment 76,000 spores ml-1, Pp blend dried spore treatment 33,500 spores ml-1, Pp3 wet spore treatment 1,500,000 spores ml-1 and Pp3 dried spore treatment 744,000 spores ml-1.
Experiment 1. Hatched J2 of M. incognita were transferred to 3.5 cm diam. Petri dishes (ca 300 J2 per dish) containing ca 10,000 spores of Pp blend to a total volume of 2.5 ml, each with five replicates, for Pp blend wet spore and Pp blend dried spore (for
2 months) treatments. After 24 h of incubation at 25-28°C, spores were observed on 10 J2 per dish in an inverted microscope at *200. The J2 encumbered with spores from each dish were used to inoculate one pepper plant (Capsicum annuum 'California Wonder') which grew in 250 ml plastic cup filled with a commercial soil substrate. Five pepper plants, inoculated with J2 free of spores incubated in distilled water to 3.5 cm diam. Petri dishes for 24 h, served as controls. Plants were kept in a growth room at 24-26°C and 14 h photoperiod for 50 days.
An efficiency of dried water suspensions of Pasteuria penetrans
Table 1. Mean number of Pasteuria penetrans (Pp) spores per second-stage juvenile (J2) of Meloidogyne spp. exposed to spore suspensions that were kept continuously wet or dried for 2 and 12 months and number of egg masses per root and number of infected females (n = 10), when spore encumbered J2 were used to inoculate plants.
Treatments Spores (J2)1 Egg masses (root) 1 Infected females (n = 10)
Exp. 1 Exp. 2 Exp. 1 Exp. 2 Exp. 1 Exp. 2
Pp blend Pp3 Pp blend Pp3 Pp blend Pp3
(M.i.) (M.j.) (M.i.) (M.j.) (M.i.) (M.j.)
2 months drying period
Control - - 20.80 a 26.2 a - -
Wet spores 5.46 a 13.26 a 4.7 b 2.0 b 8.0 a 8.6 a
Dried spores 5.58 a 15.32 a 4.8 b 2.6 b 7.6 a 8.4 a
LSD 5% 1.11 3.13 8.6 10.57 1.38 1.08
12 months drying period
Control - - 83.4 a 62.4 a - -
Wet spores 8.56 a 7.94 b 13.4 b 18.4 b 5.8 a 10.0 a
Dried spores 9.06 a 9.08 a 10.6 b 14.4 b 5.6 a 10.0 a
LSD 5% 2.14 1.04 23.75 16.77 1.26 0
Mean of five replicates per treatment; means within columns followed by the same letter are not significantly different according to LSD test (P < 0.05); 300 J2 (plant)-1 for the 2 month drying period and 1,000 J2 (plant)-1 for the 12 month drying period. Exp. 1: Meloidogyne incognita (M.i.) exposed to spores of Pp blend and inoculated on pepper; Exp. 2: M. javanica (M.j.) exposed to spores of Pp3 and inoculated on tomato.
Afterwards they were uprooted, the roots were thoroughly washed and the number of egg masses was recorded. The roots remained in water for 2 days for the tissue to be softened. Single females without egg masses in galls were observed under a stereoscope. The Pp infected females were distinguished by their dull creamy white colour (Mankau & Prasad, 1977), which sometimes had traces of gelatinous matrix without eggs inside. From each replicate plant, ten females suspected to be infected were removed and were put in drops of water on a glass slide, crushed with a cover slip and examined at ><400 for the presence of mature spores of Pp. The Pp blend spore suspension dried for 12 months and a non-treated control were used to encumber J2 (ca 1,000) in 3.5 cm diam. Petri dishes containing ca 30,000 spores to a total volume of 3 ml with five replicates for each treatment. The encumbered and spore free J2 were used to inoculate pepper plants, which were maintained in the growth room for 50 days and afterwards the number of egg masses per root was recorded and females suspected to be infected were extracted and examined, as described above.
Experiment 2. The same procedures as in Experiment 1 were repeated with Pp3 and M. javanica. The J2 were inoculated on tomato plants.
All results were analysed with single ANOVA and treatment means compared using LSD test at 5% level of significance. The analysis was conducted with the SAS University Edition.
RESULTS
The results are presented in Table 1. There are no significant differences between treatments in spore attachment on J2, number of egg masses per root and number of infected females after the 2 months drying period for both experiments. For the 12 months drying period, while there was no difference on spore attachment on J2 for Pp blend (Exp. 1), dried spores of Pp3 attached on J2 at higher rate than the wet spores (Exp. 2). Both wet spore and dried Pp for twelvemonth treatments (Exp. 1 and Exp. 2) had a similar efficacy in reducing the number of egg masses and in infection of females compared with the controls.
DISCUSSION
It has been known that spores of Pp inside dried root material lose their ability to infect females over time. A period of 6 or 11 years of storage did not affect the ability of spores to attach but significantly decreased the infection rate of females (Espanol et al., 1997; Giannakou et al., 1997). Spores of Pp stored for 24 years in the form of dried root powder attached to J2 but did not infect females (Nasiou et al., 2020). However, spores maintained in water suspensions in a domestic refrigerator for 24 years, retained their ability to infect females (Tzortzakakis, 2022).
Drying spore suspension of Pp offers the advantage of transporting the parasite for research
purposes between laboratories. Pp is a beneficial organism that occurs naturally and is harmful exclusively to plant-parasitic nematodes. However, its transportation may be blocked by posting regulations since dried root material may be considered 'suspicious' while a liquid form may be forbidden for post services. This could be overcome easily if it is in the form of a dried aqueous suspension contained in a small 'empty' vial.
The results of this work indicate that Pp spores in a water suspension, which remains dry for 2 and 12 months and hydrated afterwards, were able to attach to J2 and subsequently to infect the females. Therefore, a dried water suspension of Pp for up to 1 year can be safely used for further multiplication of the parasite.
The viability of Pp spores in dry conditions may cause problems in the laboratories with possible contamination of the infrastructure (benches, glassware etc.). It has been demonstrated that treatment of dried spores with alcohol did not prevent subsequent spore attachment, while a dilution of 0.5% commercial sodium hypochlorite, added to dried spores on a microscope slide, destroyed them (Netscher & Duponnois, 1998). Cleaning of glassware from attached spores can be achieved by autoclaving, while methods for spore elimination from non-autoclaved items (e.g., bench surface, plastic ware) should be further investigated.
REFERENCES
Chen, Z.X. & Dickson, D.W. 1998. Review of Pasteuria penetrans: biology, ecology and biological control potential. Journal of Nematology 30: 313-340. Espanol, M., Verdejo-Lucas, S., Davies, K.G. & Kerry, B.R. 1997. Compatibility between Pasteuria penetrans isolates and Meloidogyne populations from
Spain. Biocontrol Science and Technology 7: 219230. DOI: 10.1080/09583159730910 Giannakou, I.O., PEMBROKE, B. Gowen, S.R. & Davies, K.G. 1997. Effects of long-term storage and above normal temperatures on spore adhesion of Pasteuria penetrans and infection of the root-knot nematode Meloidogyne javanica. Nematologica 43: 193-198. DOI: 10.1163/004825997X00051 Hussey, R.S. & Barker, K.R. 1973. A comparison of methods of collecting inocula for Meloidogyne spp., including a new technique. Plant Disease Reporter 57: 1025-1028. Mankau, R. & Prasad, N. 1977. Infectivity of Bacillus penetrans in plant parasitic nematodes. Journal of Nematology 9: 40-45. Nasiou, E., Thoden, T., Pardavella, I.V. & Tzortzakakis, E.A. 2020. Compatibility of fluazaindolizine and oxamyl with Pasteuria penetrans on spore attachment to juveniles of Meloidogyne javanica and M. incognita. Journal of Nematology 52: 1-7. DOI: 10.21307/j ofnem-2020-070 Netscher, C. & Duponnois, R. 1998. Use of aqueous suspensions for storing and inoculating spores of Pasteuria penetrans, parasite of Meloidogyne spp. Nematologica 44: 91-94. DOI: 10.1163/ 005225998X00091 Spaull, V.W. 1981. Bacillus penetrans in South African plant-parasitic nematodes. Nematologica 27: 244-245. DOI: 10.1163/187529281X00322 STIRLING, G.R. & WACHTEL, M.F. 1980. Mass production of Bacillus penetrans for the biological control of root-knot nematodes. Nematologica 26: 308-312. DOI: 10.1163/187529280X00260 Tzortzakakis, E.A. 2022. The effect on spore viability of storing the nematode parasite Pasteuria penetrans in either dried root material or water suspension. Russian Journal of Nematology 30: 19-20. DOI: 10.24412/0869-6918-2022-1-19-20
E.A. Tzortzakakis. Влияние высушивания водной суспензии Pasteuria penetrans на жизнеспособность спор.
Резюме. Паразитирующая на галловых нематодах бактерия Pasteuria penetrans (Pp) - потенциальный агент биологической борьбы. Возможно сохранение споры бактерий в высушенных корнях, с зараженными самками мелойдогин, или в водных суспензиях измельченных зараженных самок. В связи с таможенными правилами интерес представляет высушивание самих спор, без корней и самок нематод. Высушенные водные суспензии со спорами Рр высушивали в течение двух или 12 месяцев. После гидратации высушенных спор оценивали их способность прикрепляться к личинкам M. incognita и M. javanica за 24 часа инкубации. Личинки M. incognita с прикреплёнными спорами были использованы для заражения перца, а личинки M. javanica для заражения томата. Количество яйцевых мешков и наличие спор в самках без них определяли после 50 дней выращивания в инкубационной комнате. Показана одинаковая скорость прикрепления спор и их эффективность в снижении количества яйцевых мешков и степени заражения самок, что доказывает возможность использования высушивания водных суспензий Pp для почтовой пересылки и применения этой бактерии.
