Suppression of Meloiodogyne incognita (Tylenchida: Heteroderidae) and Tylenchulus semipenterans (Tylenchida: Tylenchulidae) using Tilapia fish powder and plant growth promoting rhizobacteria in vivo and in vitro

Plant-parasitic nematodes (PPNs) in particular those belonging to the genera Meloidogyne and Tylenchulus are a limiting factor in the production of many plants. In this research, we investigate a strategy for the control of PPNs in vivo and in vitro. The purpose of this research is to evaluate different concentrations of Tilapia fish powder (TFP) and of (in the form of BECTO Grow Roots®) against egg hatching and second-stage juveniles of Meloiodogyne incognita (Tylenchida: Heteroderidae) under laboratory condition. We also assessed the effect of TFP and on M. incognita and Tylenchulus semipenterans (Tylenchida: Tylenchulidae) reproduction. Our data showed that the percentage of egg hatching inhibition ranged from 8.03 to 53.21% and 42.25 to 75.12% after five days of treatment at different concentrations of TFP and PGPR, respectively compared with the control. The percentage of M. incognita J2 mortality increase significantly (p ≤ 0.05) from 52.1 to 86.7 and 44.6 to 92.3% after seven days of treatment at different concentrations of TFP and PGPR, respectively compared with control. Under greenhouse conditions, a remarkable (p ≤ 0.05) increase in plant growth parameters was observed in cucumber plants that received TFP and In the field experiment, the highest reduction of T. semipeneterans density was TFP +  PGPR This holds both for healthy and infected trees. Healthy navel orange (HNO) + PGPR + TFP achieved maximum enhancement in orange weight/tree compared to other treatments. Our study recommended TFP and PGPR not only because of their potential against nematodes, but also because of their safety for humans, mammals and non-target organisms.


Introduction
Approximately 4000 species of plant-parasitic nematodes (PPNs) have been identified, and they may be found in all major biomes (Press & Phoenix, 2005). Plant-parasitic nematodes are parasitic worms that damage food crops including maize (Zea mays), potato (Solanum tuberosum), and tomato (Solanum lycopersicum) (Nicol et al., 2011). The global agriculture industry spends $173 billion a year on the control of PPNs (Elling, 2013). Meloidogyne incognita (Tylenchida: Heteroderidae) and Tylenchulus semipenetrans (Tylenchida: Tylenchulidae) are the most economically significant nematodes due to the high levels of damage and infection they cause, their large host range, and interaction with the host plant (Abd-Elgawad et al., 2016;Aioub et al., 2022).
Plants infected with M. incognita show symptoms both above and below ground. Aboveground, infected plants show poor growth, fewer, smaller, pale green leaves, and leaves that wilt at high temperatures. Large galls on the roots, obstruct the plant's capacity to absorb and move dissolved nutrients, are the first signs of the condition. Nematodes feed on roots, harming them and opening the door for other soilborne diseases (Agrios, 2005). Above ground T. semipenetrans symptoms are stunting, poor development, yellowing, diminished foliage and smaller fruits. The feeder roots infected with the citrus nematode are slightly thicker than healthy ones and have a dirty appearance because soil particles adhere to the gelatinous matrix deposited by the female nematode on the root surface. Infected roots reduce the tree's ability to absorb water and nutrients necessary for normal growth (Abd-Elgawad et al., 2016). Consequently, nematodes are considered one of the most dangerous pests that cause damage to crops and reduce economic production.
Indiscriminate usage of chemical nematicides bear the risk of harm to humans, animals, plants, and the ecosystem as a whole because of their a non-target impacts. Therefore, biological control of nematodes has drawn interest from researchers (Ali et al., 2022;Feng et al., 2022).
Amongst the alternative methods in the management of nematodes are the use of Tilapia fish powder (TFP) and plant growth-promoting rhizobacteria (PGPR). Using TFP is beneficial because it can reduce M. incognita population size and improve eggplant (Solanum melongena L.) growth (Kesba et al., 2021). TFP can reduc the number of M. incognita and Rotylenchulus reniformis on different strains of cowpea (Vigna unguiculata L.) under greenhouse conditions (Kesba et al., 2013). Little work has been done on the use of TFP as a material against nematodes. Plant growth-promoting rhizobacteria (PGPR) promote plant growth are host-specific biological agents that may have a negative influence on the growth and survival of PPNs (Alberton et al., 2020). Plant growth-promoting rhizobacteria are alsoknown as plant health-promoting rhizobacteria or nodulepromoting rhizobacteria (Hayat et al., 2010) and can be divided into two groups depend on their habitat, viz., iPGPR (i.e., symbiotic bacteria), which live inside plant cells, produce nodules, and are restricted to particular structures, and ePGPR (i.e., free living rhizobacteria), which exist outside plant cells, do not produce nodules, but nevertheless promote plant development (Gray & Smith, 2005). The use of PGPR in conjunction with animal manures have been suggested as aneffective, environmentally friendly method to control a variety of nematode communities and improve cucumber (Cucumis sativus) and tomato (S. lycopersicum) development might be considered as an alternative to the synthetic nematicide oxamyl (Ali et al. (2022).
The main hypothesis of this research is that TFP and PGPR might be alternatives for nematicides for the control of nematodes We evaluate TFP and PGPRefficacy against eggs and J2 of M. incognita under laboratory condition. Moreover, we assess the impact of TFP and PGPRon M. incognita reproduction on Cucumis sativus under greenhouse conditions and the reduction of T. semipenterans population density under field trials.

Preparation of Tilapia fish powder (TFP)
The Nile tilapia were collected from markets and rinsed with tap water, then dried in the oven at (60 ± 5 °C) for 3 days with removing the digestive gland and gills from the whole body before grinding with a Microhammer-mill and then sieved (2 mm mesh) (Kofoid & White) Prepared fish powders were stored at 10 °C in a sealed plastic bags until use.

PGPR source
Plant growth-promoting rhizobacteria (in the form of BECTO Grow Roots ®, involved Pseudomonas putida/and fluorescens Serratia marcescens) was obtained from the Central Laboratory of Pesticides, Dokki, Giza.
Source and inoculum of root-knot nematode,

Meloidogyne incognita
The nematode population of M. incognita was established from a single egg-mass and the culture was maintained in the greenhouse on atomato Solanum lycopersicum L., susceptible cultivar Super Strain B as a source of egg-masses, free eggs and second stage juveniles according to El-Ashry (2021). Species identification was based on measurements on second stage juvenile and examination of the perineal pattern system of adult females according to Eisenback (1985) and Jepson (1987) In vitro assay

Preparation of free eggs and second juvenile of Meloidogyne incognita
The infected roots of tomato were cut into pieces of 2 cm long and placed in a flask of volume 600 mL, containing 200 mL of 0.5% sodium hypochlorite. The flask was capped tightly and shaken for 3 min to dissolve the gelatinous matrix partially freeing eggs from the egg masses (Hussey, 1973).The liquid suspension of eggs was poured through a 200 mesh sieve nested upon a 500 mesh sieve. Eggs collected on the 500-mesh sieve were immediately washed free of residual sodium hypochlorite solution under a slow stream of tap water and incubated in Petri dishes at 25 ± 1 °C until hatching. Newly hatched juveniles were collected using a micropipette.
Egg masses of equal size needed to study the effect of the tested extracts on egg hatching of M. incognita were hand-picked with fine forceps from small galls on the infected tomato roots obtained from previously maintained pure culture. The collected egg masses were surface sterilized in 1:500 (v/v) aqueous solution of sodium hypochlorite (Clorox) for 5 min (Akhtar et al., 2005).
Extracted eggs were suspended by distilled water and counted by using a counting slide under a research microscope (100X magnification). The concentration of eggs was adjusted to about 1000 eggs per ml by diluting the suspension. Approximately 200 free eggs in 0.1 mL water were exposed to the aforementioned concentrations of Rhizobacteria or Tilapia fish powder (TFP).
Control treatment Petri dishes contained only free eggs mixed with 10 mL of distilled water only. Treatments were left under ambient temperature of 25 ± 3 °C to determine the bacterial biocontrol efficiency and TFP. All treatments were left under laboratory temperature 25 ± 3˚C and all treatments were replicated five times. Numbers of hatched juveniles were counted using a research microscope (100X magnification). The cumulative number of hatched juveniles in each Petri dish was counted after 1, 3, 5-, 7-, 10and 12-days post treatment. The percentage of hatching inhibition was calculated in comparison with the control treatment, according to the following equation: b-Second juvenile mortality The concentration of emerged juveniles was adjusted to 100 juveniles per 0.1 mL. Ten mL of PGPR or Tilapia fish powder were screened in-vitro for their biocontrol efficiency against of M. incognita at different time periods. The control treatment contained only nematodes in distilled water, and all treatments were replicated five times. Treatments were left under ambient temperature of 25 ± 3 °C to determine the bacterial biocontrol efficiency.
Tested materials were observed daily for juvenile mortality but tables only contain data of 1, 2, 3, 5, 7, 10 and 12 days as mentioned before. Juveniles showing inactive straight posture or did not show any movement after prodding were considered dead (De Nardo & Grewal, 2003). The mortality percentages were calculated as the following equation: In vivo assay

Greenhouse experiment setup and procedures
Under greenhouse conditions, a pot experiment was conducted at the faculty of agriculture, Zagazig University, Egypt. The experiment was conducted under natural light conditions. The greenhouse temperature and relative humidity were 25-27 °C and 66-69%, Mortality(%) = Dead juveniles Total number of juveniles × 100 respectively. This experiment aimed to test the efficiency of Tilapia fish powder and PGPR on cucumber (C. sativus L.) plant growth of cultivar Raian (CB898) as well as galling, and reproduction of M. incognita. Plants of C. sativus L. were transplanted into 20-cmdiameter plastic pots including 1950 g mixture of sterilized sandy soil (70.1% sand, 12.3% clay, 8.1% silt) and 120 g peat moss. The experimental pots were arranged according to a random design that consisted of eight treatments: (1) Healthy C. sativus without PGPR or Tilapia fish powder (HCS), (2) Healthy C. sativus amended with a mixture of 3 g urea and 40 mL PGPR (HCS + PGPR), (3) Healthy C. sativus amended with 10 g of Tilapia fish powder (HCS + TFP), (4) Healthy C. sativus amended with a mixture of 3 g urea and 40 mL PGPR with 10 g Tilapia fish powder (HCS + PGPR + TFP), (5) C. sativus infected with J2 of M. incognita without PGPR or TFP (CSJ), (6) C. sativus infected with J2 of M. incognita amended with a mixture of 3 g urea and 40 mL PGPR (CSJ + PGPR), (7) C. sativus infected with J2 of M. incognita amended with 10 g of Tilapia fish powder (CSJ + TFP), (8) C. sativus infected with J2 of M. incognita amended with a mixture of 3 g urea and 40 mL PGPR with 10 g Tilapia fish powder (CSJ + BECTO + TFP). 12 days after planting, (5-8) treatments were inoculated with 1000 M. incognita second stage juveniles (J2) in 2 mL water suspension, using a micropipette, at the base of the seedling via two holes 4 cm deep made using a pencil.
Two months after inoculation, plants were removed carefully from the pots and data on plant growth as indicated by length and fresh weight of shoots were recorded. Nematodes were extracted from samples of 100 g soil using a combination of sieving and Baermann trays technique (Hooper et al., 2005). Roots were soaked in tape water for one hour to remove adhering soil and to keep egg -masses on the root surface. Roots were wrapped in tissue paper to prevent drying out and the number of galls and egg masses was counted per root system. The recorded plant growth parameters included fresh root weight (g), root length (cm) and shoot weight (g). Moreover, galling and nematode development parameters including the number of egg masses/plant were assessed according to Eldeeb et al. (2022). The numbers of galls per root were enumerated and the RGI per plant root was determined using the 1-5 gall index (1 = 1-2 galls, 2 = 3-10 galls, 3 = 11-30 galls, 4 = 31-100 galls, 5 ≥ 100 galls) (Taylor & Sasser, 1978). The nematode extracts were removed, allowed to settle for 5 h, and the amount was reduced to 30 mL by getting rid of the excess (Caveness, 1975). Under the microscope, the nematode density was determined and the total number of soil nematodes per pot was computed. The reproduction factor (RF) per pot was calculated according to Sasser et al. (1984). RF = Final population/ Initial population. Host category was determined according to Canto-Saenz (1983) as follows: The percentage of changes in parameters was calculated from:

Field experiment setup and procedures
The experimental area is located in Belbes District, Sharqia Governorate, Egypt (30° 25′ 8"N, 31° 33′ 53"E). A drip irrigation system was used, and the experimental site received traditional horticulture treatments. The field experiment was conducted in navel orange treesnaturally infested by the citrus nematode, Tylenchulus semipenetrans during 2021 to test the efficiency of TFP and PGPR (35.7 L/ha of 10 8 colony-forming units/mL include S. marcescens and P. putida/P. fuorescens) against T. semipenetrans. After harvest 250 g soil and 10 g roots were collected to determine the nematode speciesWe monitored the population fluctuations of T. semipenetrans in soil samples after one, two and three months. with TFP and PGPR treatments. The experimental trees were arranged according to the following designed: (1) Navel orange infected with T. semipenetrans (Positive control) with TFP and PGPR (NOI), (2) Healthy navel orange without PGPR and TFP (Negative control) (HNO), (3) Healthy navel orange amended with a mixture of 9 mL urea and 100 mL PGPR (HNO + PGPR), (4) Healthy navel orange amended with 30 g of TFP (HNO + TFP), (5) Healthy navel orange amended with a mixture of 9 mL urea and 100 mL PGPR + and 30 g TFP (HNO + PGPR + TFP), (6) Navel orange infected with T. semipenetrans amended with a mixture of 9 mL urea + 100 mL PGPR(NOI + PGPR), (7) Navel orange infected with T. semipenetrans amended with 30 g TFP (NOI + TFP), (8) Navel orange infected with T. semipenetrans amended with a mixture of 9 mL urea and 100 mL PGPR plus 30 g TFP (NOI + PGPR + TFP). Each treatment has ten replicates. The T. semipenetrans population densities in healthy navel orange trees is around 300 individuals which below the economic threshold under field conditions. Fruit yield/trees (total weight of fruit/ trees) were evaluated at harvest time from each tested tree.

Statistical analysis
A fully randomized design was used for the laboratory experiments, whereas a randomized full block design was used for the greenhouse and field experiments. Dataanalysis was performed using MSTAT version 4, and Duncan's multiple range test was used to compare the means (p ≤ 0.05).

Results
In Vitro nematicidal effect of Tilapia fish powder and PGPR on egg hatching and juvenile mortality of M. incognita The efficacy of rhizobacteria formulated as PGPR and Tilapia fish powder (TFP) on M. incognita eggs were tested under laboratory conditions (Tables 1  and 2). Data analysis of both tested materials showed  4 showed that the effectiveness of PGPR and TFP on M. incognita J2s mortality compared with distilled water. PGPR induced higher J2 mortality (98.38%) compared toTFP 86.7% after 7 days. After 10 days of treatment with PGPR and TFP the mortality in J2 varied from 50.55% to reach 100% with PGPR concentrations of 1 × 10 5 cfu /mL and 10 8 cfu /mL compared to 60 and 96.4% with TFP concentrations of 1.0 g and 15.0 g, respectively.
Comparing Treatment 12 with Treatment 23 we see that PGPR is more effective that TFP in reducing egg hatching and TFP is more effective that PGPR in increasing J2 death rates.   that of TFP alone, with the exception of the number of egg masses per root. These trends in the individual parateters is clearly reflected in the population level parameters (Table 5). RGI and EMI values (5.0) were the highest in the CSJ treatment, whilst, the smallest values (3.0 and 3.8) were found in the CSJ+PGPR+ TFP treatment (Fig. 1). Through RGI and RF values, all infected treatments were susceptible since RGI value > 2 and RF > 1.

Impact of Tilapia fish powder and PGPR on
Tylenchulus semipeneterans population densities under field conditions Table 6 showed that all treatments significantly decreased the number of T. semipeneterans compared with control (NOI treatment). The highest reduction of T. semipeneterans number in both health and infected trees withPGPR and TFP Moreover, all treatments remarkably increased orange fruits weight PGPR was more effective in reducing T. semipenetrans population density and increasing the percentage of navel orange weight than with TFP.

Discussions
Alternative materials are widely used as eco-friendly methods for biological control of parasite pests like root-knot nematodes (Sarker et al., 2021). The result of this study showed that TFP and PGPR show a significant reduction (p ≤ 0.05) in egg hatchability and egg viability by reducing infective juveniles (IJs) of M. incognita. Also, adding PGPR and Tilapia fish powder (TFP) to the soil not only controlled M. incognita but also raised soil fertility, improved soil physical qualities to preserve production,. This is due to TFP containing ammonia, phosphorus, and orthophosphate. Anhydrous and aqueous ammonia, urea, and other ammonium compounds have all been employed directly to control nematodes, therefore ammonia plays a significant part in this process (Farahat et al., 2012). These findings agree with those of Ferial et al. (2011) who reported that phospho-fertilizer was most successful in lowering egg mass production, root galling, and egg hatching and survival of J2. Additionally, phosphate fertiliser indirectly affects nematode control by causing plants to develop systemic acquired resistance to RKN (Habash & Al-Banna, 2011). Furthermore, the effect of TFP may be attributed to nutrient content which improve plant growth and induce plant resistance to J2s infection to minimize juvenile numbers able to penetrate roots (Castro et al., 2006;Mazur & Radziemska, 2014). PGPR directly and indirectly inhibit nematodes. PGPR functions directly as biofertilizers that create organic compounds that encourage plant development by enhancing soil nutrient absorption. Whilst, indirect effects include the creation of antibiotics, Fe chelators (also known as  , and external cell wall-degrading enzymes (such as chitinase and glucanase) that may hydrolyze the cell wall of nematodes (Mhatre et al., 2019). These results are agree with (Kumar et al., 2020) who reported that one of the indirect processes is the production of 1-aminocyclopropane-1-carboxylic acid deaminase, which lowers the quantity of ethylene in plants and increases plant tolerance. HCN is a significant inhibitor of M. incognita and Agrobacterium tumefaciens,and is produced by the majority of PGPR isolates. HCNproducing rhizobacteria increased all growth parameters of tomatoes (El-Rahman et al., 2019). Therefore, extracellular secretion explained the effectiveness of the culture filtrate method for the management of RKN (Sharma & Sharma, 2017), and the quantity and type of extracellular secretion molecules determine how different PGPR isolates may be distinguished. P. putida, P. fluorescens PF19, and Bacillus subtilis isolates all have high potency (Ludwig et al., 2013;Nikoo et al., 2014;Sohrabi et al., 2018) which may depend on secondary metabolites (Viljoen et al., 2019;Zhang et al., 2022).  Under Field condition, the combination of PGPR and TFP decrease T. semipenetrans population density and increased orange yield. This may be due to TFP not only functioning as a source of inorganic nutrients, but also as a substrate for PGPR. Moreover, potential effects of TFP may be ascribed to nutritional content that enhances plant development and increases plant resistance to J2s infection (Mazur & Radziemska, 2014). Fish emulsion has previously been used as a fertilizer for plant growth (Aung and Flick 1980;Emino and ER 1981). This result is consistent with the previous report by Garcıa et al. (2005) who demonstrated that fish waste is an important source of minerals and important sources of crude protein.
Biodegradable fish waste is known to containhigh levels of dry matter, crude protein, ether extract, crude fiber, nitrogen free extract and ash as well as having a high mineral content. Additionally, fish powder alters soil pH to alkaline and it may be responsible for the reduction of plant-parasitic nematodes (Yang et al., 2016). the decomposition process of organic amendments like fish powder or fish waste converte nitrogen to ammonia that kills PPNs (Oka, 2010;Thoden et al., 2011). Akhtar & Mahmood 1995) also report that TFP decreases PPNs populations. Overall, these findings offer compelling proof that TFP and PGPR are usefullmaterials for nematode control and protect the environment from pollution.

Conclusions
This research discussed using alternatives methods to control PPNs. PGPR and Tilapia fish powder contributed significantly to the suppression of M. incognita and T. semipenetrans in vivo and in vitro. The use of PGPR and Tilapia fish powder are more environmentally friendly for controlling PPNs populations than nematicides. The combined use of PGPRand Tilapia fish powder may constitute a new biocontrol strategy for integrated pest management programs to manage PPNs. This paves the way for using environmentally safe materials (TFP and PGPR) in the management nematodes.

Data availability
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Declarations
Ethics approval and consent to participate Not applicable.

Competing interests The authors declare no competing interests.
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