Pathogenicity of reniform nematode (Rotylenchulus reniformis) on the stinking passionflower (Passiflora foetida)

Abstract Stinking passionflower (Passiflora foetida L.) is a medicinal species that may be used as rootstock to sour passion fruit (Passiflora edulis) against wilting and collar rot caused by Fusarium spp. However, as it is a host of the reniform nematode (Rotylenchulus reniformis), the cultivation of this species may be constrained in crop fields infested by this nematode. The objective of this study was to evaluate the effect of R. reniformis on the growth of stinking passion flower. The length of plants inoculated with the highest dose of each trial (152,900 and 78,900 specimens per plant corresponding to 402.4 and 207.6 specimens / cm3 of soil) was shorter than in plants not inoculated with the reniform nematode. Therefore, R. reniformis should be considered a pathogen of stinking passionflower and be properly managed.

The R. reniformis isolate was obtained in Piracicaba (SP) from sour passion fruit plants (P.edulis) with symptomatic roots and kept on the same plant in a greenhouse (25 ± 10 ºC) .The inoculum consisted of eggs and mobile stages (juveniles, males and immature females).The inoculum was obtained using a protocol adapted from Boneti and Ferraz (1981).The roots were chopped with a 1% sodium hypochlorite solution (NaClO) in a common kitchen blender (550 W) at low speed for 60 seconds.Subsequently, the resulting suspension was collected through a sieving sequence (60 -200 -500 mesh, corresponding to openings of 0.250 -0.075 -0.025 mm), removing the sodium hypochlorite with tap water.The material deposited on the 500-mesh sieve was collected in a beaker and the nematodes were counted using a Peters' slide in a compound light microscope (model CHS, Olympus Optical Co., Ltd., Tokyo, Japan) at 100x magnification.
The experiment was conducted in two trials.Trial 1 was performed using the first germinated seeds, and trial 2 was using seeds germinated later.The plantlets formed were transferred to the pots and inoculated 12 days after transplanting (Trial 1), or 21 days after transplanting (Trial 2).Both trials were performed during the summer.
Nematode doses were based on inoculum availability and in the results of Kirby (1978), which registered over 36,000 specimens of R. reniformis per 200 cm 3 of soil (180 / cm 3 ) in a very infested orchard in Fiji.Inoculation was performed by pipetting an aqueous suspension containing the nematode specimens into two holes (2 cm deep) made in the soil, dividing half of the inoculated amount between the holes.Each treatment was composed by seven replicates, each replicate corresponding to one plantlet 10-12 cm high with 5-6 leaves (trial 1) or one plantlet 12-14 cm high with 7-8 leaves (trial 2).The plants were maintained in glasshouse (25 ± 10 ºC) until the evaluations that occurred on March 7, 2022 (44 days after the inoculation in trial 1) and May 19, 2022 (56 days after the inoculation in trial 2).

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After this period of time, the soil was separated from the roots and the nematodes were recovered from it by the centrifugal flotation method proposed by Jenkins (1964) resulting in an aqueous suspension containing eggs, males and immature females.The nematodes from the roots (eggs, juveniles, immature females and males) were recovered by the adapted method of Boneti and Ferraz (1981) described previously for the production of inoculum.Finally, the nematodes were preserved alive at 10 ºC and counted twice on a Peters' counting slide with the aid of a light microscope at 100x magnification.The R. reniformis final population (Pf) was the sum of specimens from the soil and from the roots.Three plant growth variables were assessed: plant lenght (cm), root fresh weight (g) and aerial part dry weight (g).Both trials were set in a completely randomized design.
The statistical software R (R Core Team) was used for the Shapiro-Wilk test to ensure data normality and the means were compared using the Tukey honest test at the 5% significance level.
Plant length of P. foetida infected with the higher doses of R. reniformis was shorter than non-infected ones (Figure 1, Table 1).However, infected roots weighed similarly to non-infected ones.The plants inoculated with a high density of nematodes suffered damage, that triggered a greater emission of secondary roots, which could be responsible for the increase in the root volume.(Figure 1, Table 1).
Furthermore, the aerial part dry weight of infected plants did not differ from non-infected ones, which could be explained by the fact that the infected plants, being lower than the non-infected, maintained the older leaves (Figure 1, Table 1).
The present study was the first assessing the effect of the reniform nematode on the growth of stinking passionflower.Indeed, the literature only registered the effect of R. reniformis on sour passion fruit.Plants of sour passion fruit in a soil with high density of this nematode (22,750 specimens in 4,000 cm 3 soil) produced lighter fresh aerial parts than uninfected plants (30.3 versus 23.4 g), in a greenhouse trial.However, Kirby (1978) did not detect significant effects on aerial part length (129.9 versus 113.8 cm) and root fresh weight (4.9 versus 3.5 g).In another trial, the detrimental effect of R. reniformis was proved comparing the growth of P. edulis (aerial part length and weight; root weight) plants cultivated in a naturally infested soil (2,110 specimens in 1,000 cm 3 soil) with others planted in autoclaved soil (SUÁREZ;ROSALES, 2003).
Notwithstanding the large amount of egg masses observed in the roots (Figure 1), the nematode population decreased on P. foetida in both trials, probably due to the extremely high densities used, resulting in high competition for parasitism sites.This was observed in trial 1 in which the final nematode population in soil was significantly different between the nematode doses applied (Table 1).
Indeed, the susceptibility of P. foetida to R. reniformis was demonstrated previously by Paes et al. (2022) in a glasshouse trial, when the nematode density increased 16.3 times on P. foetida after 83 days of inoculation of 1,000 specimens.
In conclusion, R. reniformis should be considered pathogenic to stinking passionflower.Therefore, the cultivation of stinking passionflower as main culture or rootstock should be preceded by preventive control methods, ie.production of healthy seedlings and choice of fields free from the nematode.

Table 1 .
Effect of Rotylenchulus reniformis on the growth of Passiflora foetida and nematode reproduction, in trial 1 (44 days after inoculation) and trial 2 (56 days after the inoculation).Each value is mean of seven replicates.Means followed by the same letter in column do not differ according to Tukey test at 5% significance. *